ES3030736T3 - Cdk inhibitors and uses thereof - Google Patents

Abstract

The present disclosure provides compounds and compositions that are selective CDK inhibitors for CDK4 and/or CDK6, and methods of using them. (Automatic translation with Google Translate, no legal value)

Description

DESCRIPTION

CDK inhibitors and their uses

Background

CDK4 and CDK6 are cyclin-dependent kinases that control the transition between the G1 and S phases of the cell cycle. The S phase is the period during which the cell synthesizes new DNA and prepares to divide during mitosis. CDK4/6 activity is typically deregulated and overactive in cancer cells. Genes encoding cyclins or genes encoding CDKs themselves may be amplified or overexpressed. Additionally, the loss of endogenous CDK4 inhibitors (also known as INK4 inhibitors) through gene deletion, mutation, or promoter hypermethylation can also lead to CDK4 and CDK6 overactivity.

Attempts have been made to prepare compounds that inhibit CDK4/6 activity, and several such compounds have been disclosed in the art. For example, International Patent Publication No. WO2016015605 describes substituted 2-aminopyridine compounds that are inhibitors of cyclin-dependent kinase 4 (CDK 4) or cyclin-dependent kinase 6 (CDK 6), and that have potential utility in the treatment of inflammatory and cell proliferative diseases.

However, given the number of pathological responses that are mediated by CDK4/6, there remains a need for CDK4/6 inhibitors that can be used in the treatment of a variety of conditions, including cancer.

Summary of the invention

In one aspect, the present invention provides compounds having the structural formulas (IIb), (IIa), or (II) shown below:

or a pharmaceutically acceptable salt thereof,

in which:

R1 is alkyl; and

R2 is optionally substituted aminoalkyl; or

R1 and R2, together with the carbon atom through which they are joined, form an optionally substituted 5- or 6-membered heterocyclic ring.

Also described herein are compounds having the structure of Formula (I):

or a pharmaceutically acceptable salt thereof, in which:

X is, independently for each occurrence, halo, preferably fluoro;

XI is O or NRX1, preferably X1 is O;

RX1 is H or alkyl, preferably lower alkyl;

R1 is alkyl, preferably lower alkyl; and

R2 is optionally substituted alkyl, optionally substituted haloalkyl, optionally substituted alkenyl, optionally substituted hydroxyalkyl, optionally substituted aminoalkyl, or optionally substituted amidoalkyl; or

R1 and R2, together with the carbon atom through which they are attached, form an optionally substituted 5- or 6-membered heterocyclic ring (e.g., a pyrrolidine ring, a piperidine ring, a piperazine ring, a pyrrolidine ring, a tetrahydropyran ring, a tetrahydrofuran ring, or a morpholine ring, each of which may be optionally substituted).

Also described herein are compounds having the structure of Formula (a):

or a pharmaceutically acceptable salt thereof, in which:

X is, independently for each occurrence, halo, preferably fluoro;

R1 is alkyl, preferably lower alkyl; and

R2 is optionally substituted alkyl, optionally substituted haloalkyl, optionally substituted alkenyl, optionally substituted hydroxyalkyl, optionally substituted aminoalkyl, or optionally substituted amidoalkyl; or

R1 and R2, together with the carbon atom through which they are joined, form an optionally substituted 5- or 6-membered heterocyclic ring.

Also described herein are compounds having the structure of Formula (Ib):

or a pharmaceutically acceptable salt thereof, in which:

X is, independently for each occurrence, halo, preferably fluoro;

RX1 is H or alkyl, preferably lower alkyl; and

R2 is optionally substituted alkyl, optionally substituted haloalkyl, optionally substituted alkenyl, optionally substituted hydroxyalkyl, optionally substituted aminoalkyl, or optionally substituted amidoalkyl.

In some compounds described herein, R1 is C1-C4 alkyl (e.g., methyl, ethyl). In some compounds described herein, R2 is optionally substituted C1-C4 alkyl or (CH2)nR2a, wherein:

R2a is optionally substituted C1-C4 alkyl, optionally substituted C1-C4 haloalkyl, optionally substituted C2-C4 alkenyl, or optionally substituted C1-C4 hydroxyalkyl, optionally substituted C1-C4 alkoxy-C1-C4 alkyl, optionally substituted C1-C4 alkylamino-C1-C4 alkyl, or optionally substituted C1-C4 alkylamino-C1-C4 haloalkyl; and

n is an integer that has a value of 1 or 2.

In certain compounds described herein, R2 is optionally substituted alkyl, optionally substituted haloalkyl, optionally substituted alkenyl, optionally substituted hydroxyalkyl, or optionally substituted aminoalkyl.

For example, in certain compounds described herein, R2 is substituted C1-C4 alkyl. In other compounds, R2 is methyl, ethyl, propylenyl, n-propyl, isopropyl, n-butyl, sec-butyl, tert-butyl, (CH2)2OH, -(CH2CH(CHa))OH, (CH2)2O(CH2CHs), -(CH2)2OCH2CH3, -(CH2)2N(H)(CHs), -(CH2)2N(H)(C(CHa)a), -(CH2)2N(H)(C(O)CHa), -(CH2)2N(H)(CH2CH2F), -(CH2)2N(CH3)(CH2CH2F), -(CH2)2N(CH3)2, -(CH2)2N(CH2CH3)2, -(CH2)2N(CH2CH3)2, -(CH2CH(CH3))N(CH3)2, -CH2C(O)-NHCH3, -CH2C(0)-N(CH3)2, -CH2C(O)-N(CH2CH3)2, or -CH2C(O)-heterocyclyl, such as -heterocyclyl linked to -CH2C(O)-N.

In some compounds described herein, R2 is (CH2)nC(O)NR2aR2b or (CH2)nNR2aR2b, where:

R2a and R2b are each independently H, alkyl, haloalkyl, alkenyl, (CRcRd)mOR2e, or -C(O)alkyl; Rc, Rd, and Re are each independently H or alkyl, preferably lower alkyl;

n is an integer that has a value of 1 or 2; and

m is an integer that has a value from 2 to 5.

Alternatively, in some compounds described herein, R2 is (CH2)nC(O)NR2aR2b or (CH2)nNR2aR2b, wherein:

R2a and R2b, together with the nitrogen atom through which they are joined, form an optionally substituted 3- to 6-membered heterocyclic ring; and

n is an integer that has a value of 1 or 2.

For example, R2a and R2b, together with the nitrogen atom through which they are joined, can form an optionally substituted heterocyclic ring selected from:

in which:

Each Rab is independently halo, hydroxyl, alkyl, haloalkyl, or alkoxy;

X2 is O, NRx1 or CRx2Rx3;

Rx1, Rx2, and Rx3 are each independently H or alkyl, preferably lower alkyl; and

z is an integer that has a value from 0 to 2.

In certain compounds described herein of Formula (I) or Formula (Ia), R1 and R2, together with the carbon atom through which they are attached, form a heterocyclic ring having the structure:

in which:

X3 is NRY1ao CRY1bRY1c;

X4 is O or CRY2aRY2b;

RY1a is H, alkyl, -C(O)RY1aa; or -S(O)2alkyl;

RY1aa is alkyl or alkoxy; and

RY1b, RY1c, RY2a, and RY2b are each independently H or alkyl, preferably lower alkyl.

In some compounds described herein, the heterocyclic ring is selected from:

Also described herein are compounds of Formula (I) selected from:

or a pharmaceutically acceptable salt thereof.

Also described herein are compounds having the structure of Formula (IVa) or (IVb):

or a pharmaceutically acceptable salt thereof,

in which:

X' in each case is independently halo, preferably F;

RX1' in each case is independently H or lower alkyl;

R1' is C1-C3 alkyl;

R2' is hydroxyalkyl or (CR2c2)nNR2a'R2b';

R2a' is H, lower alkyl, acyl or haloalkyl;

R2b' is H, lower alkyl, acyl or haloalkyl; or

R2a' and R2b' together, through the N atom through which they are joined, form a 4-, 5- or 6-membered heterocyclic ring optionally substituted with Rab'z'; or

R1' and R2' together through the C atom through which they are joined, form a 5 or 6 membered heterocyclic ring optionally substituted with acyloxy;

Rab', when present, is in each occurrence independently halo, hydroxy, lower alkyl or alkoxy; each R2c' is independently H or alkyl, preferably methyl;

n' is an integer that has a value of 1 or 2 ;

z' is an integer that has a value of 0, 1, or 2; and

wherein the compound has a CDK4 Ki of about 0.960 nM or less.

Also described herein are compounds having the structure of Formula (V):

or a pharmaceutically acceptable salt thereof,

in which:

R3a and R3b, taken together with the nitrogen atom to which they are attached, form an optionally substituted spirocyclic moiety [3.3], wherein the optionally substituted spirocyclic moiety [3.3] optionally comprises at least one additional heteroatom selected from O, S, and SO2, provided that the compound is not

Compounds having the structure of Formula (Va) are also described herein:

or a pharmaceutically acceptable salt thereof,

in which:

R3a’ and R3b’, taken together with the nitrogen atom to which they are attached, form a structure selected from:

in which

Each Rab is independently halo, hydroxyl, alkyl, haloalkyl, or alkoxy; and

z is 0, 1, or 2.

In the compounds described herein, R3a’ and R3b’, taken together with the nitrogen atom to which they are attached, form

each Rab is independently halo; and

z is 2.

In the compounds described herein, R3a’ and R3b’, taken together with the nitrogen atom to which they are attached, form

in which Rab is fluorine.

Also described herein are compositions (e.g., pharmaceutical compositions) comprising a compound of Formula (I), Formula (IVa), Formula (IVb), Formula (V), or Formula (Va), and a carrier (e.g., a pharmaceutically acceptable carrier, such as a diluent or excipient). Also described herein are methods of treating a condition or disorder in a subject in need thereof, comprising administering to the subject a compound of Formula (I), Formula (Ia), Formula (Ib), Formula (IVa), Formula (IVb), Formula (V), or Formula (Va), or a pharmaceutically acceptable salt thereof. Such conditions and disorders include, but are not limited to, cancers, viral infections, inflammatory diseases, cardiovascular diseases, neurodegenerative disorders, glomerulonephritis, myelodysplasia syndromes, myocardial infarctions associated with ischemic injury, stroke and reperfusion injury, arrhythmia, atherosclerosis, toxin-induced or alcohol-related liver diseases, hematological diseases, degenerative diseases of the musculoskeletal system, and ophthalmic diseases.

Also described herein are methods of sensitizing cancer and/or tumor cells in a subject in need thereof to a chemotherapeutic agent or radiation comprising administering to the subject a CDK4 and/or CDK inhibitor in an amount sufficient to arrest the cancer and/or tumor cell cycle, and thereby sensitizing the cancer and/or tumor cells in the mammal to a chemotherapeutic agent or radiation, wherein the CDK4 and/or CDK6 inhibitor is a compound of Formula (I), Formula (Ia), Formula (Ib), Formula (IVa), Formula (IVb), Formula (V) or Formula (Va), or a pharmaceutically acceptable salt thereof.

Also described herein are methods for inhibiting CDK4 and/or CDK6 in a cell comprising contacting said cell with a compound of Formula (I), Formula (Ia),

Formula (Ib), Formula (IVa), Formula (IVb), Formula (V) or Formula (Va), or a pharmaceutically acceptable salt thereof, such that the CDK4 and/or CDK6 enzymes are inhibited in said cell.

Brief description of the drawings

FIG. 1 shows the IC50 distribution of Abemaciclib by cancer type.

FIG. 2 shows the IC50 distribution of compound A1 (mesylate salt) by cancer type.

FIG. 3 shows the IC50 distribution of compound A22 (mesylate salt) by cancer type.

FIG. 4 shows the IC50 distribution of compound A23 (mesylate salt) by cancer type.

FIG. 5 shows the IC50 distribution of compound A2 (mesylate salt) by cancer type.

FIG. 6 shows the relative selectivity of the CDK4/6 inhibitors palbociclib (200 nM), ribociclib (200 nM), and abemaciclib (200 nM). Compared with palbociclib and ribociclib, abemaciclib targets additional kinases: CDK16, CDK7, DYRK1B, GSK3B, JNK1/2/3, PIM1, ROCK2, and PRKCE; ribociclib is the cleanest, but targets ULK2.

FIG. 7 shows a kinase inhibition screen of certain compounds at 200 nM compared to abemaciclib at 200 nM.

FIG. 8 shows a kinase inhibition screen of certain compounds at 2000 nM compared to abemaciclib at 2000 nM.

FIG. 9 shows a single-dose PK experiment at 8.3 mg/kg exploring PO versus IP and comparative exposure to abemaciclib. This exploratory PK study in mice suggested variations in the PK of the tested compounds. For example, PO-administered compounds A1 and A2 have low Cmax, with slow metabolism and a longer half-life compared to abemaciclib. PO-administered compound A49 has a high Cmax, followed by rapid metabolism. IP-administered compounds A1 and A2 achieved similar bioavailability to abemaciclib.

FIGS. 10 and 11 show efficacy data from the first ER+ breast cancer cell line xenograft study over 26 days of dosing with certain compounds of the invention. Compound A1 was administered IP at 60 mg/kg, with a 2-day rest period, and restarted at 20 mg/kg QD from day 5 onward. Compound A22 was administered PO and increased to 120 mg/kg from day 12 onward. Compound A49 was administered PO and dosing was discontinued on day 19. The ZR751 cell line was used for this study.

FIGS. 12A and 12B show the results of the first ER+ breast cancer cell line xenograft study.

FIG. 13 shows the results of a follow-up PK study for certain compounds of the invention, each dosed at 100 mg/kg PO.

FIG. 14 shows an example treatment plan for the 2nd ER+ breast cancer cell line xenograft study. The ZR751 cell line was used for this study.

FIGS. 15 and 16 show the results of the second xenograft efficacy study at day 22.

FIG. 17 shows the PK results of the second xenograft efficacy study, which were determined from plasma collected at 240 and 1,440 minutes postdose on day 10 of the study.

FIG. 18A shows comparative AUC measurements for Compounds A2, A1, A23, and abemaciclib at doses of 100 to 1000 mg/kg.

FIG. 18B shows comparative PK curves for Compounds A2, A1, A23, and abemaciclib at doses of 100 to 1000 mg/kg.

FIG. 19 shows an example design of a 14-day dose tapering study.

FIGS. 20 and 21 show the results of a 14-day dose-taper study for Compound A2. The maximum tolerated dose for Compound A2 was between about 278 and about 399 mg/kg QD.

FIGS. 22 and 23 show the results of a 14-day dose-taper study for Compound A1. The maximum tolerated dose for Compound A1 was less than approximately 400 mg/kg QD.

FIGS. 24 and 25 show the results of a 14-day dose-escalation study for Compound A23. The maximum tolerated dose for Compound A23 was approximately 200 mg/kg QD. FIGS. 26 and 27 show the results of a 14-day dose-escalation study for bemaciclib. The maximum tolerated dose for abemaciclib was between approximately 100 and approximately 150 mg/kg QD.

FIG. 28 shows an example treatment plan for a xenograft study involving 8 mice per experimental group. Blood draws were taken on the first and last days of treatment.

FIGS. 29 and 30 show xenograft efficacy data during the 26-day study and on the final day (day 26), respectively.

FIG. 31 is a graphical representation of animal body weight data during the 26-day xenograft study.

FIG. 32 shows the PK data of Compounds A1, A2, A23, and abemaciclib in the day 25 blood draw of the xenograft study.

FIG. 33 shows the accumulation of compounds of the invention versus abemaciclib during a 26-day xenograft study.

FIG. 34 tabulates body weight data during the 25 days of dosing in the xenograft study. FIGS. 35A and 35B tabulate animal body weight data on day 26 (final day) of the xenograft study.

FIG. 36 shows a summary of the results of the xenograft efficacy study.

FIG. 37 shows a summary of the logarithmic differences in the average IC50 for certain compounds in resistant cell lines and sensitive cell lines.

Detailed description of the invention

Abemaciclib (brand name Verzenio™):

inhibits the enzymes cyclin-dependent kinase 4 (CDK4) and cyclin-dependent kinase 6 (CDK6). These enzymes are responsible for phosphorylating and thus activating the retinoblastoma protein, which plays a role in cell cycle progression from the G1 phase (first gap) to the S phase (synthesis). Blocking this pathway prevents cells from progressing to the S phase, which induces apoptosis (cell death). The compounds disclosed herein share certain structural features of abemaciclib and may also act as selective CDK inhibitors for CDK4 and/or CDK6.

Definitions

Unless otherwise defined herein, the scientific and technical terms used in this application shall have the meanings commonly understood by those of ordinary skill in the art. In general, the nomenclature used in connection with, and the techniques of, chemistry, cell and tissue culture, molecular biology, cell and cancer biology, neurobiology, neurochemistry, virology, immunology, microbiology, pharmacology, genetics, and protein and nucleic acid chemistry described herein are those well known and commonly used in the art.

The methods and techniques of the present disclosure are generally performed, unless otherwise indicated, in accordance with conventional methods well known in the art and as described in various general and more specific references cited and discussed throughout this specification. See, e.g., “Principles of Neural Science,” McGraw-Hill Medical, New York, N.Y. (2000); Motulsky, “Intuitive Biostatistics,” Oxford University Press, Inc. (1995); Lodish et al., “Molecular Cell Biology, 4th ed.,” W. H. Freeman & Co., New York (2000); Griffiths et al., “Introduction to Genetic Analysis, 7th ed.,” W. H. Freeman & Co., N.Y. (1999); and Gilbert et al., “Developmental Biology, 6th ed.,” Sinauer Associates, Inc., Sunderland, MA (2000).

Chemical terms used herein, unless otherwise defined, are used in accordance with conventional usage in the art as exemplified in “The McGraw-Hill Dictionary of Chemical Terms”, Parker S., Ed., McGraw-Hill, San Francisco, CA (1985).

All of the foregoing, and any other publications, patents, and published patent applications referenced in this application, are specifically incorporated by reference herein. In the event of a conflict, this specification, including its specific definitions, shall prevail.

A “patient,” “subject,” or “individual” are used interchangeably and refer to a human or a nonhuman animal. These terms include mammals, such as humans, primates, livestock animals (including bovines, porcines, etc.), companion animals (e.g., canines, felines, etc.), and rodents (e.g., mice and rats).

“Treating” a condition or a patient refers to taking steps to achieve beneficial or desired outcomes, including clinical outcomes. As used herein, and as is well understood in the art, “treatment” is an approach to achieving beneficial or desired outcomes, including clinical outcomes. Beneficial or desired clinical outcomes may include, but are not limited to, the alleviation or amelioration of one or more symptoms or conditions, the slowing of disease extent, stabilization (i.e., non-worsening) of the disease state, prevention of disease spread, delay or slowing of disease progression, improvement or palliation of the disease state, and remission (whether partial or complete), whether detectable or undetectable. “Treatment” can also mean prolonging survival compared to expected survival if untreated.

The term “preventing” is recognized in the art and when used in connection with a condition, such as a local recurrence (e.g., pain), a disease such as cancer, a syndrome complex such as heart failure, or any other medical condition, is well understood in the art, and includes administering a composition that reduces the frequency of, or delays the onset of symptoms of a medical condition in a subject relative to a subject not receiving the composition. Thus, preventing cancer includes, for example, reducing the number of detectable cancerous growths in a patient population receiving prophylactic treatment relative to an untreated control population, and/or delaying the occurrence of detectable cancerous growths in a treated population versus an untreated control population, for example, by a statistically and/or clinically significant amount.

“Administering” or “administering” a substance, compound, or agent to a subject can be carried out using one of a variety of methods known to those skilled in the art. For example, a compound or agent can be administered intravenously, arterially, intradermally, intramuscularly, intraperitoneally, subcutaneously, ocularly, sublingually, orally (by ingestion), intranasally (by inhalation), intraspinally, intracerebrally, and transdermally (by absorption, e.g., through a cutaneous conduit). A compound or agent can also be appropriately introduced by refillable or biodegradable polymeric devices or other devices, e.g., patches and pumps, or formulations, that provide for extended, slow, or controlled release of the compound or agent. Administration can also be performed, e.g., once, a plurality of times, and/or over one or more extended periods.

Appropriate methods for administering a substance, compound, or agent to a subject will also depend, for example, on the age and/or physical condition of the subject and the chemical and biological properties of the compound or agent (e.g., solubility, digestibility, bioavailability, stability, and toxicity). In some embodiments, a compound or agent is administered orally, for example, to a subject by ingestion. In some embodiments, the orally administered compound or agent is in an extended-release or slow-release formulation, or is administered using a device for such extended-release or slow-release.

As used herein, the phrase “co-administration” refers to any form of administration of two or more different therapeutic agents such that the second agent is administered while the previously administered therapeutic agent is still effective in the body (e.g., both agents are simultaneously effective in the patient, which may include synergistic effects of the two agents). For example, the different therapeutic compounds may be administered in the same formulation or in separate formulations, either concomitantly or sequentially. In this way, an individual receiving such treatment may benefit from a combined effect of different therapeutic agents.

The term “acyl” is art recognized and refers to a group represented by the general formula hydrocarbylC(O)-, preferably alkylC(O)-.

The term “acylamino” is art-recognized and refers to an amino group substituted with an acyl group and may be represented, for example, by the hydrocarbyl formula C(O)NH-.

The term “acyloxy” is art recognized and refers to a group represented by the general formula hydrocarbylC(O)O-, preferably alkylC(O)O-.

The term “alkoxy” refers to an alkyl group, preferably a lower alkyl group, having an oxygen bonded thereto. Representative alkoxy groups include methoxy, trifluoromethoxy, ethoxy, propoxy, tert-butoxy, and the like.

The term “alkoxyalkyl” refers to an alkyl group substituted with an alkoxy group and can be represented by the general formula alkyl-O-alkyl.

The term “alkenyl,” as used herein, refers to an aliphatic group containing at least one double bond and is intended to include both “unsubstituted alkenyls” and “substituted alkenyls,” the latter of which refers to alkenyl moieties having substituents replacing a hydrogen on one or more carbons of the alkenyl group. Such substituents may appear on one or more carbons that are either included or not included in one or more double bonds. Furthermore, such substituents include all those contemplated for alkyl groups, as discussed below, except where stability is prohibitive. For example, substitution of alkenyl groups with one or more alkyl, carbocyclyl, aryl, heterocyclyl, or heteroaryl groups is contemplated.

An “alkyl” or “alkane” group is a non-aromatic, straight- or branched-chain hydrocarbon that is fully saturated. Typically, a straight- or branched-chain alkyl group has from 1 to about 20 carbon atoms, preferably from 1 to about 10, unless otherwise defined. Examples of straight- and branched-chain alkyl groups include methyl, ethyl, n-propyl, iso-propyl, n-butyl, sec-butyl, tert-butyl, pentyl, hexyl, pentyl, and octyl. A C1-C6 straight- or branched-chain alkyl group is also referred to as a “lower alkyl” group.

Furthermore, the term “alkyl” (or “lower alkyl”) as used throughout the specification, examples and claims is intended to include both “unsubstituted alkyls” and “substituted alkyls,” the latter of which refers to alkyl moieties having substituents replacing a hydrogen on one or more carbons of the hydrocarbon backbone. Such substituents, unless otherwise specified, may include, for example, a halogen (e.g., fluoro), a hydroxyl, a carbonyl (such as a carboxyl, an alkoxycarbonyl, a formyl, or an acyl), a thiocarbonyl (such as a thioester, a thioacetate, or a thioformate), an alkoxy, a phosphoryl, a phosphate, a phosphonate, a phosphinate, an amino, an amido, an amidine, an imine, a cyano, a nitro, an azido, a sulfhydryl, an alkylthio, a sulfate, a sulfonate, a sulfamoyl, a sulfonamido, a sulfonyl, a heterocyclyl, an aralkyl, or an aromatic or heteroaromatic moiety. In preferred embodiments, substituents on substituted alkyls are selected from C1-C6 alkyl, C3-C6 cycloalkyl, halogen, carbonyl, cyano, or hydroxyl. In more preferred embodiments, substituents on substituted alkyls are selected from fluoro, carbonyl, cyano, or hydroxyl. Those skilled in the art will understand that substituted groups on the hydrocarbon chain may themselves be substituted, if appropriate. For example, substituents on a substituted alkyl can include substituted and unsubstituted forms of amino, azido, imino, amido, phosphoryl (including phosphonato and phosphinato), sulfonyl (including sulfate, sulfonamido, sulfamoyl, and sulfonato), and silyl groups, as well as ethers, alkylthios, carbonyls (including ketones, aldehydes, carboxylates, and esters), -CF3, -CN, and the like. Exemplary substituted alkyls are described below. Cycloalkyls may be further substituted with alkyls, alkenyls, alkoxys, alkylthios, aminoalkyls, carbonyl-substituted alkyls, -CF3, -CN, and the like.

The term “Cx-Cy,” when used in conjunction with a chemical moiety such as acyl, acyloxy, alkyl, alkenyl, alkynyl, or alkoxy, is intended to include groups containing x to y carbons in the chain. For example, the term “Cx-Cy alkyl” refers to substituted or unsubstituted saturated hydrocarbon groups, including straight-chain alkyl groups and branched-chain alkyl groups containing x to y carbons in the chain, including haloalkyl groups. Preferred haloalkyl groups include trifluoromethyl, difluoromethyl, 2,2,2-trifluoroethyl, and pentafluoroethyl. C<q>alkyl denotes a hydrogen where the group is in a terminal position, a bond if internal. The terms “C2-Cy alkenyl” and “C2-Cy alkynyl” refer to substituted or unsubstituted unsaturated aliphatic groups analogous in length and possible substitution to the alkyls described above, but containing at least one double or triple bond, respectively.

The term “alkylamino” as used herein refers to an amino group substituted with at least one alkyl group.

The term “alkylthio” as used herein refers to a thiol group substituted with an alkyl group and may be represented by the general formula alkylS-.

The term “alkynyl,” as used herein, refers to an aliphatic group containing at least one triple bond and is intended to include both “unsubstituted alkynyls” and “substituted alkynyls,” the latter of which refers to alkynyl moieties having substituents replacing a hydrogen on one or more carbons of the alkynyl group. Such substituents may appear on one or more carbons that are either included or not included in one or more triple bonds. Moreover, such substituents include all those contemplated for alkyl groups, as discussed above, except where stability is prohibitive. For example, substitution of alkynyl groups with one or more alkyl, carbocyclyl, aryl, heterocyclyl, or heteroaryl groups is contemplated.

The term “amide” as used herein refers to a group

in which each RA independently represents a hydrogen or hydrocarbyl group, or two RAs are taken together with the N atom to which they are attached and complete a heterocycle that has from 4 to 8 atoms in the ring structure.

The terms “amine” and “amino” are art-recognized and refer to both substituted and unsubstituted amines and salts thereof. For example, a moiety that may be represented by

in which each RA independently represents a hydrogen or hydrocarbyl group, or two RAs are taken together with the N atom to which they are attached and complete a heterocycle having from 4 to 8 atoms in the ring structure.

The term “aminoalkyl” as used herein refers to an alkyl group substituted with an amino group.

The term “aryl” as used herein includes substituted or unsubstituted single ring aromatic groups in which each ring atom is carbon. Preferably, the ring is a 6 or 10 membered ring, more preferably a 6 membered ring. The term “aryl” also includes polycyclic ring systems having two or more cyclic rings in which two or more carbons are common to two adjacent rings in which at least one of the rings is aromatic, for example, the other cyclic rings may be cycloalkyls, cycloalkenyls, cycloalkynyls, aryls, heteroaryls and/or heterocyclyls. Aryl groups include benzene, naphthalene, phenanthrene, phenol, aniline and the like.

A “cycloalkyl” group is a cyclic hydrocarbon that is fully saturated. “Cycloalkyl” includes both monocyclic and bicyclic rings. Typically, a monocyclic cycloalkyl group has from 3 to about 10 carbon atoms, more usually from 3 to 8 carbon atoms unless otherwise defined. The second ring of a bicyclic cycloalkyl may be selected from saturated, unsaturated, and aromatic rings. Cycloalkyl includes bicyclic molecules in which one, two, or three or more atoms are shared between the two rings (e.g., fused bicyclic compounds, bridged bicyclic compounds, and spirocyclic compounds).

The term “fused bicyclic compound” refers to a bicyclic molecule in which two rings share two adjacent atoms. In other words, the rings share a covalent bond, i.e., the so-called bridgehead atoms are directly connected (e.g., α-thujene and decalin). For example, in a fused cycloalkyl, each of the rings shares two adjacent atoms with the other ring, and the second ring of a fused bicyclic cycloalkyl can be selected from saturated, unsaturated, and aromatic rings. A “cycloalkenyl” group is a cyclic hydrocarbon containing one or more double bonds.

The term “bridged bicyclic compound” refers to a bicyclic molecule in which the two rings share three or more atoms, with the two bridgehead atoms separated by a bridge containing at least one atom. For example, norbornane, also known as bicyclo[2.2.1]heptane, can be thought of as a pair of cyclopentane rings that each share three of their five carbon atoms.

The term “spirocyclic compound” refers to a bicyclic molecule in which the two rings have only one atom, the spiro atom, in common.

The terms “halo” and “halogen” as used herein mean halogen and include chlorine, fluorine, bromine and iodine.

The term “heteroalkyl,” as used herein, refers to a saturated or unsaturated chain of carbon atoms and at least one heteroatom, in which no two heteroatoms are adjacent.

The terms “heteroaryl” and “hetaryl” include substituted or unsubstituted aromatic single ring structures, preferably 5- to 7-membered rings, more preferably 5- to 6-membered rings, which ring structures include at least one heteroatom, preferably one to four heteroatoms, more preferably one or two heteroatoms. The terms “heteroaryl” and “hetaryl” also include polycyclic ring systems having two or more cyclic rings in which two or more carbons are common to two adjacent rings in which at least one of the rings is heteroaromatic, for example, the other cyclic rings may be cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl and/or heterocyclyl. Heteroaryl groups include, for example, pyrrole, furan, thiophene, imidazole, oxazole, thiazole, pyrazole, pyridine, pyrazine, pyridazine, and pyrimidine, and the like.

The term “heteroatom” as used herein means an atom of any element other than carbon or hydrogen. Preferred heteroatoms are nitrogen, oxygen, and sulfur.

The terms “heterocyclyl”, “heterocycle” and “heterocyclic” refer to substituted or unsubstituted non-aromatic ring structures, preferably 3 to 10 membered rings, more preferably 3 to 7 membered rings, which ring structures include at least one heteroatom, preferably one to four heteroatoms, more preferably one or two heteroatoms. The terms “heterocyclyl” and “heterocyclic” also include polycyclic ring systems having two or more cyclic rings in which two or more carbons are common to two adjacent rings in which at least one of the rings is heterocyclic, for example, the other cyclic rings may be cycloalkyls, cycloalkenyls, cycloalkynyls, aryls, heteroaryls and/or heterocyclyls. Heterocyclyl groups include, for example, pyrrolidine, piperidine, piperazine, pyrrolidine, tetrahydropyran, tetrahydrofuran, morpholine, lactones, lactams and the like.

The term “hydroxyalkyl,” as used herein, refers to an alkyl group substituted with a hydroxy group.

The term “lower” when used in conjunction with a chemical moiety, such as acyl, acyloxy, alkyl, alkenyl, alkynyl, or alkoxy, is intended to include groups in which there are ten or fewer non-hydrogen atoms in the substituent, preferably six or fewer. A “lower alkyl,” for example, refers to an alkyl group containing ten or fewer carbon atoms, preferably six or fewer. In certain embodiments, the acyl, acyloxy, alkyl, alkenyl, alkynyl, or alkoxy substituents defined herein are respectively lower acyl, lower acyloxy, lower alkyl, lower alkenyl, lower alkynyl, or lower alkoxy, whether appearing alone or in combination with other substituents, such as in the mentions hydroxyalkyl and aralkyl (in which case, for example, the atoms within the aryl group are not counted when counting the carbon atoms in the alkyl substituent).

The term “substituted” refers to moieties having substituents replacing a hydrogen on one or more carbons in the principal chain. “Substitution” or “substituted with” shall be understood to include the implied condition that such substitution be in accordance with the permissible valency of the substituted atom and the substituent, and that the substitution results in a stable compound, e.g., one that does not spontaneously undergo a transformation such as rearrangement, cyclization, elimination, etc. As used herein, the term “substituted” is contemplated to include all permissible substituents of organic compounds. In a broad aspect, permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and non-aromatic substituents of organic compounds. Permissible substituents may be one or more and the same or different for the appropriate organic compounds. For the purposes of this invention, heteroatoms such as nitrogen may have hydrogen substituents and/or any permissible substituents of the organic compounds described herein that satisfy the valencies of the heteroatoms. Substituents may include any substituents described herein, for example, a halogen, a hydroxyl, a carbonyl (such as a carboxyl, an alkoxycarbonyl, a formyl, or an acyl), a thiocarbonyl (such as a thioester, a thioacetate, or a thioformate), an alkoxy, a phosphoryl, a phosphate, a phosphonate, a phosphinate, an amino, an amido, an amidine, an imine, a cyano, a nitro, an azido, a sulfhydryl, an alkylthio, a sulfate, a sulfonate, a sulfamoyl, a sulfonamido, a sulfonyl, a heterocyclyl, an aralkyl, or an aromatic or heteroaromatic moiety. In preferred embodiments, the substituents on substituted alkyls are selected from C1-C6 alkyl, C3-C6 cycloalkyl, halogen, carbonyl, cyano, or hydroxyl. In more preferred embodiments, the substituents on substituted alkyls are selected from fluoro, carbonyl, cyano, or hydroxyl. Those skilled in the art will understand that the substituents may be substituted, if appropriate. Unless specifically indicated as “unsubstituted,” references to chemical moieties herein are understood to include substituted variants. For example, reference to an “aryl” group or moiety implicitly includes substituted and unsubstituted variants.

“Protecting group” refers to a group of atoms that, when attached to a reactive functional group in a molecule, masks, reduces, or prevents the reactivity of the functional group. Typically, a protecting group can be selectively removed as desired during the course of a synthesis. Examples of protecting groups can be found in Greene and Wuts, Protective Groups in Organic Chemistry, 3rd Ed., 1999, John Wiley & Sons, NY, and Harrison et al., Compendium of Synthetic Organic Methods, Vols. Representative nitrogen protecting groups include, but are not limited to, formyl, acetyl, trifluoroacetyl, benzyl, benzyloxycarbonyl (“CBZ”), tert-butoxycarbonyl (“Boc”), trimethylsilyl (“TMS”), 2-trimethylsilylethanesulfonyl (“TES”), trityl and substituted trityl, allyloxycarbonyl, 9-fluorenylmethyloxycarbonyl (“FMOC”), nitro-veratryloxycarbonyl (“NVOC”), and the like. Representative hydroxyl protecting groups include, but are not limited to, those in which the hydroxyl group is acylated (esterified) or alkylated such as benzyl and trityl ethers, as well as alkyl ethers, tetrahydropyranyl ethers, and trialkylsilyl ethers. (e.g. TMS or TIPS groups), glycol ethers, such as ethylene glycol and propylene glycol derivatives and allyl ethers.

The term “modulate” as used herein includes the inhibition or suppression of a function or activity (such as cell proliferation) as well as the enhancement of a function or activity.

The phrase “pharmaceutically acceptable” is art-recognized. In certain embodiments, the term includes compositions, excipients, adjuvants, polymers, and other materials and/or dosage forms that, within the scope of sound medical judgment, are suitable for use in contact with human and animal tissues without undue toxicity, irritation, allergic response, or other problems or complications, commensurate with a reasonable benefit/risk ratio.

“Pharmaceutically acceptable salt” or “salt” is used herein to refer to an acid addition salt or a base addition salt that is suitable or compatible with the treatment of patients.

The term “pharmaceutically acceptable acid addition salt” as used herein means any non-toxic organic or inorganic salt of any of the base compounds disclosed herein. Illustrative inorganic acids that form suitable salts include hydrochloric, hydrobromic, sulfuric, and phosphoric acids, as well as metal salts such as sodium monohydrogen orthophosphate and potassium hydrogen sulfate. Illustrative organic acids that form suitable salts include mono-, di-, and tricarboxylic acids such as glycolic, lactic, pyruvic, malonic, succinic, glutaric, fumaric, malic, tartaric, citric, ascorbic, maleic, benzoic, phenylacetic, cinnamic, and salicylic acids, as well as sulfonic acids such as p-toluenesulfonic and methanesulfonic acids. Mono- or diacid salts may be formed, and such salts may exist in hydrated, solvated, or substantially anhydrous forms. In general, the acid addition salts of the compounds disclosed herein are more soluble in water and various hydrophilic organic solvents, and generally exhibit higher melting points compared to their free base forms. The selection of the appropriate salt will be known to one of skill in the art. Other non-pharmaceutically acceptable salts, e.g., oxalates, may be used, for example, in the isolation of compounds of the invention for laboratory use or for subsequent conversion to a pharmaceutically acceptable acid addition salt.

The term “pharmaceutically acceptable base addition salt” as used herein means any non-toxic organic or inorganic base addition salt of any acidic compounds of the invention, or any intermediates thereof. Illustrative inorganic bases that form suitable salts include lithium, sodium, potassium, calcium, magnesium, or barium hydroxide. Illustrative organic bases that form suitable salts include aliphatic, alicyclic, or aromatic organic amines such as methylamine, trimethylamine, and picoline or ammonia. The selection of the appropriate salt will be known to one of skill in the art.

Many of the compounds useful in the methods and compositions of this disclosure have at least one stereogenic center in their structure. This stereogenic center may be present in an R or S configuration, said R and S notation being used in correspondence with the rules described in Pure Appl. Chem. (1976), 45, 11-30. The disclosure contemplates all stereoisomeric forms, such as enantiomeric and diastereomeric forms of the compounds, salts, prodrugs, or mixtures thereof (including all possible mixtures of stereoisomers). See, e.g., WO 01/062726.

Furthermore, certain compounds containing alkenyl groups may exist as Z (combined) or E (individual) isomers. In each case, the disclosure includes both mixtures and separate individual isomers.

Some of the compounds may also exist in tautomeric forms. Such forms, although not explicitly indicated in the formulas described herein, are intended to be included within the scope of this disclosure.

“Prodrug” or “pharmaceutically acceptable prodrug” refers to a compound that is metabolized, e.g., hydrolyzed or oxidized, in the host after administration to form the compound of the present disclosure (e.g., compounds of the invention). Typical examples of prodrugs include compounds that have biologically labile or cleavable (protecting) groups on a functional moiety of the active compound. Prodrugs include compounds that can be oxidized, reduced, aminated, deaminated, hydroxylated, dehydroxylated, hydrolyzed, dehydrolyzed, alkylated, dealkylated, acylated, deacylated, phosphorylated, or dephosphorylated to produce the active compound. Examples of prodrugs utilizing ester or phosphoramidate as biologically labile or cleavable (protecting) groups are described in U.S. Patents 6,875,751, 7,585,851, and 7,964,580, the disclosures of which are incorporated herein by reference. The prodrugs of this disclosure are metabolized to produce a compound of the invention, or a pharmaceutically acceptable salt thereof. The present disclosure includes within its scope, prodrugs of the compounds described herein. Conventional procedures for the selection and preparation of suitable prodrugs are described, for example, in “Design of Prodrugs” Ed. H. Bundgaard, Elsevier, 1985.

The phrase “pharmaceutically acceptable carrier” as used herein means a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filter, diluent, excipient, solvent or encapsulating material useful in formulating a drug product for medicinal or therapeutic use.

As used herein, “a CDK4/6 inhibitor” or “CDK4/6 inhibitor therapy” refers to a compound or composition that inhibits the activity of CDK4/6, for example, by phosphorylating a serine or threonine residue in proteins, or inhibits the interaction of CDK4/6 with other proteins that may be in the signal pathway.

As used herein, “cyclin-dependent kinase 4/6 (CDK4/6) inhibitor-sensitive” or “CDK4/6-sensitive cancer” refers to a cell or cancer that has reduced growth in the presence of a CDK4/6 inhibitor compared to the absence of such inhibitor. Sensitivity may refer to a cytotoxic or cytostatic effect of the CDK4/6 inhibitor on the cell. It is contemplated that a sensitive cell line may have a 1.5-, 2-, 3-, 4-, 5-, 6-, 7-, 8-, 9-, 10-, 15-, 20-, 25-fold or greater change in growth rate in the presence of a CDK4/6 inhibitor. Sensitivity may also be measured by change in genome sequence or gene copy number, increase or decrease in expression of a particular protein or mRNA expression, or other measurement disclosed herein as a measure of sensitivity.

The term “response to CDK4 and/or CDK6 inhibitors” refers to any response of a hyperproliferative disorder (e.g., cancer) to an agent that inhibits CDK4 or CDK6, preferably a change in tumor mass and/or volume after initiation of chemotherapy. Response to a hyperproliferative disorder may be assessed, for example, to evaluate efficacy or in a neoadjuvant or adjuvant setting, where the size of a tumor after a systemic intervention can be compared to the initial size and dimensions as measured by CT, PET, mammography, ultrasound, or palpation. Responses may also be assessed by caliper measurement or pathologic examination of the tumor after biopsy or surgical resection. Response may be recorded quantitatively, such as percentage change in tumor volume, or qualitatively, such as “pathological complete response” (pCR), “clinical complete remission” (cCR), “clinical partial remission” (cPR), “clinical stable disease” (cSD), “clinical progressive disease” (cPD), or other qualitative criteria. Assessment of response to the hyperproliferative disorder may be performed shortly after the start of neoadjuvant or adjuvant therapy, for example, after a few hours, days, weeks, or, preferably, after a few months. A typical endpoint for response assessment is the completion of neoadjuvant chemotherapy or surgical removal of residual tumor cells and/or the tumor bed. This typically occurs three months after the start of neoadjuvant therapy. In some embodiments, the clinical efficacy of the therapeutic treatments described herein may be determined by measuring the clinical benefit rate (CBR). The clinical benefit rate is measured by determining the sum of the percentage of patients who are in complete remission (CR), the number of patients who are in partial remission (PR), and the number of patients who have stable disease (SD) at a time at least 6 months after the end of therapy. The abbreviation for this formula is CBR=CR+PR+SD for 6 months. In some embodiments, the CBR for a particular cancer therapeutic regimen is at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or more. Additional criteria for evaluating response to cancer therapies are related to “survival,” which includes all of the following: survival to mortality, also known as overall survival (where such mortality may be independent of cause or tumor-related); “recurrence-free survival” (where the term “recurrence” includes both localized and distant recurrence); metastasis-free survival; disease-free survival (where the term “disease” includes cancer and diseases associated with it). The duration of such survival may be calculated with reference to a defined starting point (e.g., time of diagnosis or start of treatment) and end point (e.g., death, recurrence, or metastasis). Furthermore, treatment efficacy criteria may be extended to include response to chemotherapy, probability of survival, probability of metastasis within a given time period, and probability of tumor recurrence. For example, to determine appropriate threshold values, a particular anticancer therapeutic regimen may be administered to a population of subjects, and the outcome may be correlated with biomarker measurements that were determined prior to the administration of any anticancer therapy. The outcome measure may be the pathological response to the therapy administered in the neoadjuvant setting. Alternatively, outcome measures, such as overall survival and disease-free survival, can be monitored over a period of time for subjects following cancer therapy for whom biomarker measurement values are known. In certain embodiments, the doses administered are standard doses known in the art for cancer therapeutic agents. The period of time for which subjects are monitored may vary. For example, subjects may be monitored for at least 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 45, 50, 55, or 60 months.

The term “survival” includes all of the following: survival to mortality, also known as overall survival (where such mortality may be independent of cause or related to a tumor); “recurrence-free survival” (where the term “recurrence” includes both localized and distant recurrence); metastasis-free survival; disease-free survival (where the term “disease” includes cancer and diseases associated with it). The duration of such survival may be estimated with reference to a defined starting point (e.g., time of diagnosis or start of treatment) and end point (e.g., death, recurrence, or metastasis). In addition, criteria for treatment efficacy may be extended to include response to chemotherapy, probability of survival, probability of metastasis within a given time period, and probability of tumor recurrence.

As used herein, “cyclin-dependent kinase 4/6 (CDK4/6) inhibitor-resistant” or “CDK4/6-resistant cancer” refers to a cell or cancer that has normal (or reference) growth in the presence of a CDK4/6 inhibitor and is substantially similar to that in the absence of such inhibitor. Resistance can be measured by a relative maintenance of the cell growth rate in the presence of a CDK4/6 inhibitor, or by a change in the genome sequence or copy number of a gene, an increase or reduction in the expression of a particular protein or in mRNA expression, or other measurement disclosed herein as a measure of resistance.

The term “sensitize” means to alter cancer cells or tumor cells in a manner that allows for more effective treatment of the cancer associated with a cancer therapy (e.g., immune checkpoint therapy, chemotherapy, and/or radiation therapy). In some embodiments, normal cells are not affected to the extent that they would be unduly harmed by the immune checkpoint therapy. Increased sensitivity or reduced sensitivity to a therapeutic treatment is measured according to a method known in the art for the particular treatment and the methods described below, including, but not limited to, cell proliferation assays (Tanigawa N, Kern D H, Kikasa Y, Morton D L, Cancer Res 1982; 42: 2159-2164), and cell death assays (Weisenthal L M, Shoemaker R H, Marsden J A, Dill P L, Baker J A, Moran E M, Cancer Res 1984; 94: 161-173; Weisenthal L M, Lippman M E, Cancer Treat Rep 1985; 69: 615-632; Weisenthal L M, In: Kaspers G J L, Pieters R, Twentyman P R, Weisenthal L M, Veerman A J P, eds. Drug Resistance in Leukemia and Lymphoma. Langhorne, P A: Harwood Academic Publishers, 1993: 415-432; Weisenthal L M, Contrib Gynecol Obstet 1994; 19: 82-90). Sensitivity or resistance can also be measured in an animal by measuring the reduction in tumor size over a period of time, e.g., 6 months for a human and 4-6 weeks for a mouse. A composition or method sensitizes the response to a therapeutic treatment if the increase in sensitivity to the treatment or reduction in resistance is 25% or more, e.g., 30%, 40%, 50%, 60%, 70%, 80% or more, to 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 15-fold, 20-fold or more, compared to the sensitivity or resistance to the treatment in the absence of such composition or method. Determining sensitivity or resistance to a therapeutic treatment is standard practice in the art and is within the skill of a skilled artisan. It should be understood that any method described herein for enhancing the efficacy of a cancer therapy is equally applicable to methods for sensitizing hyperproliferative or otherwise cancerous cells (e.g., resistant cells) to cancer therapy.

Compounds of the invention

The compounds of the present invention have the structure of Formula (IIb), (IIa), or (II) shown below:

or a pharmaceutically acceptable salt thereof, in which:

R1 is alkyl; and

R2 is optionally substituted aminoalkyl; or

R1 and R2, together with the carbon atom through which they are joined, form an optionally substituted 5- or 6-membered heterocyclic ring.

In one embodiment, R1 is C1-C4 alkyl, preferably methyl or ethyl. In a further embodiment, R2 is -(CH2)2N(H)(CH3), -(CH2)2N(H)(C(CH3)3),-(CH2)2N(H)(CH2CH2F), -(CH2)2N(CH3)(CH2CH2F), -(CH2)2N(CH3)2, -(CH2)2N(CH2CH3)2, -(CH2)2N(CH2CH3)2, and -(CH2CH(CH3))N(CH3)2. In a further embodiment, R2 is (CH2)nNR2aR2b, wherein: R2a and R2b are each independently H, alkyl, haloalkyl, alkenyl, or (CRcRd)mORe; Rc, Rd, and Re are each independently H or alkyl; n is an integer that has a value of 1 or 2; and m is an integer that has a value from 2 to 5.

Particular compounds of the invention are selected from:

or a pharmaceutically acceptable salt thereof.

In a particular embodiment of the invention, the compounds have the structure of Formula (IVa):

or a pharmaceutically acceptable salt thereof,

in which:

X' is fluorine;

R1' is C1-C3 alkyl;

R2' is (CR2c2)n NR2a'R2b';

R2a' is H, lower alkyl or haloalkyl;

R2b' is H, lower alkyl or haloalkyl

each R2c' is independently H or alkyl;

n' is an integer that has a value of 1 or 2; and

wherein the compound has a CDK4 Ki of about 0.960 nM or less.

Additional compounds of the invention are selected from:

or a pharmaceutically acceptable salt thereof.

Compounds of disclosure

Described herein are compounds having the structure of Formula (I):

or a pharmaceutically acceptable salt thereof, in which:

X1 is O or NRX1;

RX1 is H or alkyl, preferably lower alkyl;

R1 is alkyl, preferably lower alkyl; and

R2 is optionally substituted alkyl, optionally substituted haloalkyl, optionally substituted alkenyl, optionally substituted hydroxyalkyl, optionally substituted aminoalkyl, or optionally substituted amidoalkyl; or

R1 and R2, together with the carbon atom through which they are joined, form an optionally substituted 5- or 6-membered heterocyclic ring.

In certain compounds described herein, R2 is optionally substituted alkyl, optionally substituted haloalkyl, optionally substituted alkenyl, optionally substituted hydroxyalkyl, or optionally substituted aminoalkyl.

Also described herein are compounds having the structure of Formula (Ia):

or a pharmaceutically acceptable salt thereof, in which:

X is, independently for each occurrence, halo, preferably fluoro;

R1 is alkyl, preferably lower alkyl; and

R2 is optionally substituted alkyl, optionally substituted haloalkyl, optionally substituted alkenyl, optionally substituted hydroxyalkyl, optionally substituted aminoalkyl, or optionally substituted amidoalkyl; or

R1 and R2, together with the carbon atom through which they are joined, form an optionally substituted 5- or 6-membered heterocyclic ring.

In certain compounds described herein, R2 is optionally substituted alkyl, optionally substituted haloalkyl, optionally substituted alkenyl, optionally substituted hydroxyalkyl, or optionally substituted aminoalkyl.

Also described herein are compounds having the structure of Formula (Ib):

or a pharmaceutically acceptable salt thereof, in which:

X is, independently for each occurrence, halo, preferably fluoro;

RX1 is H or alkyl, preferably lower alkyl; and

R2 is optionally substituted alkyl, optionally substituted haloalkyl, optionally substituted alkenyl, optionally substituted hydroxyalkyl, optionally substituted aminoalkyl or optionally substituted amidoalkyl.

In certain compounds described herein, R2 is optionally substituted alkyl, optionally substituted haloalkyl, optionally substituted alkenyl, optionally substituted hydroxyalkyl, or optionally substituted aminoalkyl.

Also described herein are compounds of Formula (I) having the structure of Formula (II):

or a pharmaceutically acceptable salt thereof.

Also described herein are compounds of Formula (II) having the structure of Formula (IIa):

or a pharmaceutically acceptable salt thereof. Alternatively, compounds of Formula (II) have the structure of Formula (IIb):

or a pharmaceutically acceptable salt thereof.

Also described herein are compounds of Formula (I) having the structure of Formula (III):

or a pharmaceutically acceptable salt thereof.

Also described herein are compounds of Formula (III) having the structure of Formula (IIIa):

or a pharmaceutically acceptable salt thereof. Alternatively, compounds of Formula (III) have the structure of Formula (IIIb):

or a pharmaceutically acceptable salt thereof.

In certain compounds described herein of Formulas (I), (Ia), (Ib), (III), (IIIa) and (IIIb), RX1 is H or methyl. In preferred compounds RX1 is H.

In certain compounds described herein, R1 is C1-C4 alkyl. In some of these compounds, R1 is methyl or ethyl. In certain compounds, R1 is methyl.

In certain compounds described herein, R2 is optionally substituted C1-C4 alkyl or (CH2)nR2a, wherein R2a is optionally substituted C1-C4 alkyl, optionally substituted C1-C4 haloalkyl, optionally substituted C2-C4 alkenyl, or optionally substituted C1-C4 hydroxyalkyl, C1-C4 alkoxy-optionally substituted C1-C4 alkyl, C1-C4 alkylamino-optionally substituted C1-C4 alkyl, or C1-C4 alkylamino-optionally substituted C1-C4 haloalkyl; and n is an integer having a value of 1 or 2. In certain compounds described herein, R2 is substituted C1-C4 alkyl. In certain other compounds described herein, R2 is methyl, ethyl, propylenyl, n-propyl, isopropyl, n-butyl, sec-butyl, tert-butyl, (CH2)2OH, -(CH2CH(CH3))OH, (CH2)2O(CH2CH3), -(CH2)2OCH2CH3, -(CH2)2N(H)(CH3), -(CH2)2N(H)(C(CH3)3), -(CH2)2N(H)(C(O)CH3), -(CH2)2N(H)(CH2CH2F), -(CH2)2N(CH3)(CH2CH2F), (CH2)2N(CHa)2, -(CH2)2N(CH2CHs)2, -(CH2)2N(CH2CH3)2, -(CH2CH(CHa))N(CHa)2, -CH2C(O)-NHCH3, -CH2C(O)-N(CH3)2, -CH2C(O)-N(CH2CH3)2, or -CH2C(O)-heterocyclyl, such as heterocyclyl linked to -CH2C(O)-N-. In certain other compounds described herein, R2 is methyl, ethyl, propylenyl, n-propyl, isopropyl, n-butyl, sec-butyl, tert-butyl, (CH2)2OH, -(CH2CH(CH3))OH, (CH2)2O(CH2CH3), -(CH2)2OCH2CH3, -(CH2)2N(H)(CH3), -(CH2)2N(H)(C(CH3)3), -(CH2)2N(H)(C(O)CH3), -(CH2)2N(H)(CH2CH2F), -(CH2)2N(CH3) (CH2CH2F), -(CH2)2N(CH3)2, -(CH2)2N(CH2CH3)2, -(CH2)2N(CH2CH3)2, or -(CH2CH(CH3))N(CH3)2.

In other compounds described herein, R2 is (CH2)nC(O)NR2aR2b, where R2a and R2b are each independently H, alkyl, haloalkyl, alkenyl, (CRcRd)mORe, or -C(O)alkyl; Rc, Rd, and Re are each independently H or alkyl, preferably lower alkyl; n is an integer having a value of 1 or 2; and m is an integer having a value of 2 to 5.

In other compounds described herein, R2 is (CH2)nNR2aR2b, where R2a and R2b are each independently H, alkyl, haloalkyl, alkenyl, (CRcRd)mORe, or -C(O)alkyl; Rc, Rd, and Re are each independently H or alkyl, preferably lower alkyl; n is an integer having a value of 1 or 2; and m is an integer having a value of 2 to 5.

In still other compounds described herein, R2 is (CH2)nC(O)NR2aR2b, wherein R2a and R2b, together with the nitrogen atom through which they are attached, form an optionally substituted 3- to 6-membered heterocyclic ring; and n is an integer having a value of 1 or 2. In some such compounds described herein, R2a and R2b, together with the nitrogen atom through which they are attached, form an optionally substituted heterocyclic ring selected from:

in which:

Each Rab is independently halo, hydroxyl, alkyl, haloalkyl, or alkoxy;

X2 is O, NRx1 or CRx2Rx3;

Rx-i, rx2, and rx3 are each independently H, halo, alkyl, preferably lower alkyl, or alkoxy; and

z is an integer that has a value from 0 to 2.

In still other certain compounds described herein, R2 is (CH2)nNR2aR2b, wherein R2a and R2b, together with the nitrogen atom through which they are attached, form an optionally substituted 3- to 6-membered heterocyclic ring; and n is an integer having a value of 1 or 2. In some such compounds described herein, R2a and R2b, together with the nitrogen atom through which they are attached, form an optionally substituted heterocyclic ring selected from:

in which:

Each Rab is independently halo, hydroxyl, alkyl, haloalkyl, or alkoxy;

X2 is O, NRx1 or CRx2Rx3;

Rx-i,<r>x2, and<r>x3 are each independently H, halo, alkyl, preferably lower alkyl, or alkoxy; and

z is an integer that has a value from 0 to 2.

For example, the optionally substituted heterocyclic ring may be selected from:

In some compounds described herein, R1 and R2, together with the carbon atom through which they are attached, form a heterocyclic ring having the structure:

in which:

X3 is NRY1ao CRY1bRY1c;

X4 is O CRY2aRY2b;

RY1a is H, alkyl, -C(O)RY1aa; or -S(O)2alkyl;

RY1aa is alkyl or alkoxy; and

RY1b, RY1c, RY2a, and RY2b are each independently H or alkyl, preferably lower alkyl.

In some of said compounds described herein, the heterocyclic ring is selected from:

and

In some of the compounds described herein,

X1 is O or NRX1;

RX1 is H or alkyl, preferably lower alkyl;

R1 is methyl; and

R2 is optionally substituted alkyl, optionally substituted hydroxyalkyl, or (CR2c2)nNR2aR2b; or R1 and R2, together with the carbon atom through which they are joined, form a 5- or 6-membered heterocyclic ring having one N atom and the N atom is optionally substituted with lower alkyl;

R2a is H, methyl, or ethyl;

R2b is H, methyl, or ethyl;

each R2c is independently H or alkyl, preferably methyl; and

n is an integer that has a value from 1 to 4.

In some compounds described herein,

X1 is O or NRX1;

RX1 is H, methyl or ethyl;

R1 is methyl;

R2 is (CR2c2)nNR2aR2b;

R2a is H, methyl, or ethyl;

R2b is H, methyl, or ethyl;

each R2c is independently H or alkyl, preferably methyl; and

n is an integer that has a value from 1 to 4.

In some certain compounds described herein, each R2c is H. In other compounds described herein, at least one R2c is alkyl, preferably methyl, and the remainder are H. In certain compounds described herein, X1 is O. In some compounds described herein, (CR2c2)nNR2aR2b, wherein at least one R2c is optionally alkyl and the remainder are H. In other compounds described herein, wherein X1 is NRX1. In some certain compounds described herein, (CR2c2)nNR2aR2b, wherein at least one R2c is optionally methyl and the remainder are H.

In the particular compounds described herein, R2a and R2b are not both H.

In some of the compounds described herein,

X1 is O;

R1 is methyl;

R2 is optionally substituted hydroxyalkyl or optionally substituted C1-C4 alkyl-NHR2a, where R2a is methyl or ethyl; or

R1 and R2, together with the carbon atom through which they are joined, form a 5- or 6-membered heterocyclic ring having an N atom optionally substituted with lower alkyl.

In some of the compounds described herein,

X1 is O or NRX1;

RX1 is H or alkyl, preferably lower alkyl;

R1 is methyl or ethyl;

R2 is lower alkyl, (CH2)nOH or (CR2c2)nNR2aR2b; or

R1 and R2, together with the carbon atom through which they are joined, form a 5- or 6-membered heterocyclic ring having one N atom substituted with -C(O)oxyalkyl;

R2a is H or lower alkyl optionally substituted with one or more halogens;

R2b is H or lower alkyl optionally substituted with one or more halogens; and

R2a and R2b together through the N atom through which they are joined, form a 3, 4 or 5 membered heterocyclic ring optionally substituted with Rabz, in which:

Rab is halogen, hydroxyl, lower alkyl, haloalkyl, oxyalkyl;

each R2c is independently H or alkyl, preferably methyl;

z is an integer that has a value from 0 to 2; and

n is an integer that has a value from 2 to 4.

In some compounds described herein, R2a and R2b are not both H.

In some compounds described herein,

X1 is O or NRX1;

RX1 is H or alkyl, preferably lower alkyl;

R1 is methyl;

R2 is C1-C2 alkyl or (CH2)nNR2aR2b; or

R1 and R2, together with the carbon atom through which they are joined, form a 5- or 6-membered heterocyclic ring having an N atom optionally substituted with -C(O)alkyl;

R2a is unsubstituted lower alkyl;

R2b is unsubstituted lower alkyl;

n is an integer that has a value from 2 to 4.

In some compounds described herein,

X1 is O or NRX1;

RX1 is H or alkyl, preferably lower alkyl;

R1 is methyl;

R2 is C1-C2 alkyl or (CR2c2)nNR2aR2b;

R2a is unsubstituted lower alkyl;

R2b is unsubstituted lower alkyl;

each R2c is independently H or alkyl, preferably methyl; and

n is an integer that has a value from 2 to 4.

In some compounds described herein,

X1 is O or NRX1;

RX1 is H or alkyl, preferably lower alkyl; R1 is alkyl, preferably lower alkyl;

R2 is C1-C3 alkyl, C1-C3 alkenyl, optionally substituted hydroxyalkyl, optionally substituted alkoxyalkyl, or (CR2c2)nNR2aR2b; or

R1 and R2, together with the carbon atom through which they are joined, form a 5- or 6-membered heterocyclic ring having one heteroatom selected from N and O and is optionally substituted with lower alkyl, carbonyl, tert-butyloxycarbonyl, -C(O)oxyalkyl, or -S(O)2alkyl;

R2a and R2b are each independently H, alkyl, or -C(O)alkyl; or

R2a and R2b together through the N atom through which they are joined, form a 3- to 6-membered heterocyclic ring optionally having one C replaced with O, wherein the heterocyclic ring is optionally substituted with (Rab)z,

each Rab is independently halogen, hydroxyl, or lower alkyl; each R2c is independently H or alkyl, preferably methyl;

z is an integer that has a value of 1 or 2;

n is an integer that has a value from 2 to 4.

In certain compounds described herein, the compound of Formula (I) is selected from:

5

5

5

5

or a pharmaceutically acceptable salt thereof.

In certain compounds described herein, the compound of Formula (I) is selected from:

5

5

5

5

and

or a pharmaceutically acceptable salt thereof.

In some compounds described herein, the compound of Formula (I) is selected from:

or a pharmaceutically acceptable salt thereof.

In certain preferred compounds described herein, the compound of Formula (I) is selected from:

or a pharmaceutically acceptable salt thereof.

In other preferred compounds described herein, the compound of Formula (I) is selected from:

or a pharmaceutically acceptable salt thereof.

In still other preferred compounds described herein, the compound of Formula (I) is selected from:

or a pharmaceutically acceptable salt thereof.

In still other preferred compounds described herein, the compound of Formula (I) is selected from

or a pharmaceutically acceptable salt thereof.

In other preferred compounds described herein, the compound of Formula (I) is selected from:

and

or a pharmaceutically acceptable salt thereof.

In still other preferred compounds described herein, the compound of Formula (I) is selected from:

or a pharmaceutically acceptable salt thereof.

In still other preferred compounds described herein, the compound of Formula (I) is selected from

or a pharmaceutically acceptable salt thereof.

In other preferred compounds described herein, the compound of Formula (I) is selected from:

or a pharmaceutically acceptable salt thereof.

In some compounds described herein, the compound of Formula (I) is selected from:

or a pharmaceutically acceptable salt thereof.

In some compounds described herein, the compound is a mesylate salt.

In certain compounds described herein, the compound has the structure of Formula (IVa) or (IVb):

or a pharmaceutically acceptable salt thereof,

in which:

X' in each case is independently halo, preferably F;

Rx1' in each case is independently H or lower alkyl;

R1' is C1-C3 alkyl;

R2' is hydroxyalkyl or (CR2c2)nNR2a'R2b';

R2a' is H, lower alkyl, acyl or haloalkyl;

R2b' is H, lower alkyl, acyl or haloalkyl; or

R2a' and R2b' together, through the N atom through which they are joined, form a 4-, 5- or 6-membered heterocyclic ring optionally substituted with Rab'z'; or

R1' and R2' together through the C atom through which they are joined, form a 5 or 6 membered heterocyclic ring optionally substituted with acyloxy;

Rab’, when present, is in each occurrence independently halo, hydroxy, lower alkyl or alkoxy; each R2c’ is independently H or alkyl, preferably methyl;

n’ is an integer that has a value of 1 or 2;

z’ is an integer that has a value of 0, 1, or 2; and

wherein the compound has a CDK4 Ki of about 0.960 nM or less.

In some compounds described herein, the compound of Formula (IVa) or (IVb) has an average IC50 of 150 nM or less for the drug-sensitive cell lines of Table 2. In certain compounds described herein, the average IC50 of the compound of Formula (IVa) or (IVb) for the drug-sensitive cell lines of Table 2 is at least about 5-fold more potent than the average IC50 of the same compound of Formula (IVa) or (IVb) for the drug-resistant cell lines of Table 2.

In certain compounds described herein, the compound of Formula (IVa) or (IVb) has a Papp A to B score of about 0.07 or greater.

In certain compounds described herein, the compound of Formula (IVa) or (IVb) has a half-life of about 25 minutes or greater.

In some compounds described herein, the compound of Formula (IVa) or (IVb) is selected from:

, or a pharmaceutically acceptable salt thereof.

In some compounds described herein, the compound has the structure of Formula (V):

or a pharmaceutically acceptable salt thereof,

in which:

R3a and R3b, taken together with the nitrogen atom to which they are attached, form an optionally substituted spirocyclic moiety [3.3], wherein the optionally substituted spirocyclic moiety [3.3] optionally comprises at least one additional heteroatom selected from O, S, and SO2,

as long as the compound is not

In some compounds described herein, the compound has the structure of Formula (Va):

or a pharmaceutically acceptable salt thereof,

in which:

R3a’ and R3b’, taken together with the nitrogen atom to which they are attached, form a structure selected from:

in which

Each Rab is independently halo, hydroxyl, alkyl, haloalkyl, or alkoxy; and

z is 0, 1, or 2.

In certain compounds described herein, R3a’ and R3b’, taken together with the nitrogen atom to which they are attached, form

each Rab is independently halo; and

z is 2.

In some compounds described herein, R3a’ and R3b’, taken together with the nitrogen atom to which they are attached, form

where Rab is fluorine.

In certain compounds described herein, the compound is selected from:

or a pharmaceutically acceptable salt thereof.

Treatment methods

The compounds of the present invention and described herein are inhibitors of CDK4/6 and may therefore be useful for treating diseases in which the underlying pathology is (at least in part) mediated by CDK4/6. Such diseases include cancer and other diseases in which there is a disorder of cell proliferation, apoptosis, or differentiation.

Examples of cancers that can be treated with a compound of the present invention include, but are not limited to, carcinoma, for example, a carcinoma of the bladder, breast, colon (for example, colorectal carcinomas such as colon adenocarcinoma and colon adenoma), kidney, epidermis, liver, lung (for example, adenocarcinoma, small cell lung cancer, and non-small cell lung carcinomas), esophagus, gallbladder, ovary, pancreas (for example, exocrine pancreatic carcinoma), stomach, cervix, thyroid, nose, head and neck, prostate, and skin (for example, squamous cell carcinoma). Other examples of cancers that can be treated with a compound of the present invention include hematopoietic tumors of lymphoid lineage (e.g., leukemia, acute lymphocytic leukemia, mantle cell lymphoma, chronic lymphocytic leukemia, B-cell lymphoma (such as diffuse large B-cell lymphoma), T-cell lymphoma, multiple myeloma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, hairy cell lymphoma, and Burkett's lymphoma; hematopoietic tumors of myeloid lineage, e.g., acute and chronic myelogenous leukemias, myelodysplastic syndrome, and promyelocytic leukemia. Other cancers include follicular thyroid cancer; a tumor of mesenchymal origin, e.g., fibrosarcoma or habdomyosarcoma; a tumor of the central or peripheral nervous system, e.g., astrocytoma, neuroblastoma, glioma, or schwannoma; melanoma; seminoma; teratocarcinoma; osteosarcoma; xeroderma pigmentosum; retinoblastoma; keratoctomantoma; follicular thyroid cancer; and Kaposi's sarcoma.

One group of cancers includes human breast cancers (e.g., primary breast tumors, lymph node negative breast cancer, invasive ductal adenocarcinomas of the breast, non-endometrioid breast cancers) and endometrial cancers. Another subset of cancers in which compounds having CDK4/6 inhibitory activity may be of particular therapeutic benefit include glioblastoma multiforme, T-cell ALL, sarcomas, familial melanoma, and melanoma. CDK4/6 inhibitors may also be useful in the treatment of viral infections, e.g., herpesvirus, smallpox virus, Epstein-Barr virus, Sindbis virus, adenovirus, HIV, HPV, hCv, and HCMV; prevention of the development of AIDS in HIV-infected individuals; Chronic inflammatory diseases, e.g., systemic lupus erythematosus, autoimmune-mediated glomerulonephritis, rheumatoid arthritis, psoriasis, inflammatory bowel disease, and autoimmune diabetes mellitus; cardiovascular diseases, e.g., cardiac hypertrophy, restenosis, atherosclerosis; neurodegenerative disorders, e.g., Alzheimer's disease, AIDS-related dementia, Parkinson's disease, amyotrophic lateral sclerosis, retinitis pigmentosa, spinal muscular atrophy, and cerebellar degeneration; glomerulonephritis; myelodysplasia syndromes, myocardial infarctions associated with ischemic injury, stroke, and reperfusion injury, arrhythmia, atherosclerosis, toxin-induced or alcohol-related liver diseases, hematological diseases, e.g., chronic anemia and aplastic anemia; Degenerative diseases of the musculoskeletal system, for example, osteoporosis and arthritis, aspirin-sensitive rhinosinusitis, cystic fibrosis, multiple sclerosis, kidney diseases, ophthalmic diseases including age-related macular degeneration, uveitis, and cancer pain.

Furthermore, the compounds of the present invention may be useful in the treatment of tumors with amplifications of the CDK4 and CDK6 genes, as well as tumors that overexpress cyclin partners of cyclin-dependent kinases. In particular, the compounds of the present invention may be useful in the treatment of RB+ve (retinoblastoma protein positive) tumors, which include tumors harboring mutations in Ras, Raf, Growth Factor Receptors, or overexpression of Growth Factor Receptors. In addition, the compounds of the present invention may also be useful in the treatment of RB-ve tumors.

The compounds of the present invention may also be useful in the treatment of tumors with genetic aberrations that activate CDK4/6 kinase activity. These include, but are not limited to, cancers with D-cyclin translocations, such as mantle cell lymphoma and multiple myeloma, D-cyclin amplifications such as breast cancer and squamous cell esophageal cancer, CDK4 amplifications such as liposarcoma, CDK6 amplifications or overexpressions such as T-cell lymphoma, and p16 inactivation such as melanoma, non-small cell lung cancer, and pancreatic cancer. The compounds of the present invention may be useful in the treatment of cancers that have genetic aberrations in upstream regulators of D-cyclins, where the defect results in an increased abundance of D-cyclin, may also be considered for treatment. These include, but are not limited to, acute myeloid leukemia with FLT3 activation, breast cancers with Her2/neu overexpression, ER dependence or triple-negative phenotype, colon cancers with activating mutations of the MAPK, Pl3K or WNT pathway, melanomas with activating mutations of the MAPK pathway, non-small cell lung cancers with activating aberrations of the EGFR pathway, and pancreatic cancers with activating aberrations of the MAPK pathway, including K-ras mutations.

The treatment methods described herein comprise administering a compound of the invention, or a pharmaceutically acceptable salt thereof, to a subject in need thereof. Individual methods described herein include methods for treating any one of the aforementioned disorders or diseases by administering an effective amount of a compound of the invention, or a pharmaceutically acceptable salt thereof, to a subject in need thereof.

The pharmaceutical composition or combination of the present invention may be in a unit dosage of about 1-1000 mg of active ingredient(s) for a subject of about 50-70 kg, or about 1-500 mg, or about 1-250 mg, or about 1-150 mg, or about 0.5-100 mg, or about 1-50 mg of active ingredients. The therapeutically effective dosage of a compound, pharmaceutical composition, or combinations thereof depends upon the subject's species, body weight, age, and individual condition, disorder or disease, or severity thereof being treated. A physician, clinician, or veterinarian of ordinary skill can readily determine the effective amount of each of the active ingredients necessary to prevent, treat, or inhibit the progress of the disorder or disease. The aforementioned dosage properties are demonstrable in in vitro and in vivo tests advantageously utilizing mammals, e.g., mice, rats, dogs, monkeys, or isolated organs, tissues, and preparations thereof. The compounds of the present invention can be administered in vitro in the form of solutions, for example, aqueous solutions, and in vivo either by enteral, parenteral, advantageously intravenous, for example, as a suspension or in aqueous solution. The in vitro dosage may vary between about 10-9 molar and 10-3 molar concentrations. A therapeutically effective amount in vivo may vary, depending on the route of administration, between about 0.1-500 mg/kg, between about 1-100 mg/kg or between about 100-300 mg/kg.

Also described herein is a method of modulating CDK4/6 activity in a subject comprising administering to the subject a compound of the invention, or a pharmaceutically acceptable salt thereof. Additionally, described herein is a method for treating a CDK4/6-mediated disorder or disease in a subject in need thereof, comprising administering to the subject the compound of formula (I), (Ia), (Ib), (IVa), (IVb), (V), or (Va), or a pharmaceutically acceptable salt thereof. Also described herein is a method of treating a CDK4/6-mediated disorder or disease, in a subject in need of treatment thereof, comprising administering a compound of the invention, or a pharmaceutically acceptable salt thereof, wherein the disorder or disease is selected from carcinomas with genetic aberrations that activate CDK4/6 kinase activity. These include, but are not limited to, cancers with D-cyclin translocations such as mantle cell lymphoma and multiple myeloma, D-cyclin amplifications such as breast cancer and squamous cell esophageal cancer, CDK4 amplifications such as liposarcoma, CDK6 amplifications or overexpressions such as T-cell lymphoma, and p16 inactivation such as melanoma, non-small cell lung cancer, and pancreatic cancer.

Also described herein is the use of a compound of the invention, or a pharmaceutically acceptable salt thereof, for the treatment of a CDK4-mediated disorder or disease.

Also described herein is a compound of the invention, or a pharmaceutically acceptable salt thereof, for use in the treatment of a CDK4-mediated disorder or disease, in a subject wherein the disorder or disease is selected from carcinomas with genetic aberrations that activate CDK4/6 kinase activity. These include, but are not limited to, cancers with D-cyclin translocations such as mantle cell lymphoma and multiple myeloma, D-cyclin amplifications such as breast cancer and squamous cell esophageal cancer, CDK4 amplifications such as liposarcoma, CDK6 amplifications or overexpressions such as T-cell lymphoma, and p16 inactivation such as melanoma, non-small cell lung cancer, and pancreatic cancer.

Also described herein is a compound according to Formula (I), (Ia), (Ib), (IVa), (IVb), (V) or (Va), or a pharmaceutically acceptable salt thereof, for use as a medicament.

Also described herein is the use of a compound of Formula (I), (Ia), (Ib), (IVa), (IVb), (V) or (Va), or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for the treatment of a CDK4/6 mediated disorder or disease, wherein the disorder or disease is selected from carcinomas with genetic aberrations that activate CDK4/6 kinase activity. These include, but are not limited to, cancers with D-cyclin translocations such as mantle cell lymphoma and multiple myeloma, D-cyclin amplifications such as breast cancer and squamous cell esophageal cancer, CDK4 amplifications such as liposarcoma, CDK6 amplifications or overexpressions such as T-cell lymphoma and p16 inactivation such as melanoma, non-small cell lung cancer and pancreatic cancer.

Combinations

The compounds of the present invention may be administered concomitantly, either simultaneously with, or before or after, one or more other therapeutic agents. The compounds of the present invention may be administered separately, by the same or different route of administration, or together in the same pharmaceutical composition as the other agents.

In some embodiments, the invention provides a product comprising a compound of the invention, or a pharmaceutically acceptable salt thereof, and at least one other therapeutic agent as a combined preparation for simultaneous, separate, or sequential use in therapy. In some such products, the therapy is the treatment of a disease or condition mediated by CDK4/6 inhibition. Products provided as a combined preparation include a composition comprising the compound of the invention and the other therapeutic agent(s) together in the same pharmaceutical composition, or the compound of the invention and the other therapeutic agent(s) in separate form, e.g., in kit form. In certain embodiments, the invention provides a pharmaceutical composition comprising a compound of the invention, or a pharmaceutically acceptable salt thereof, and other therapeutic agent(s). Optionally, the pharmaceutical composition may comprise a pharmaceutically acceptable excipient, as described above.

Also described herein is a kit comprising two or more separate pharmaceutical compositions, at least one of which contains a compound of the invention or a pharmaceutically acceptable salt thereof. In some such kits, the kit comprises means for separately storing said compositions, such as a container, a divided bottle, or a divided foil packet. An example of such a kit is a blister pack, such as those typically used for packaging tablets, capsules, and the like.

The kit can be used to administer different dosage forms, for example, oral and parenteral, to administer the separate compositions at different dosage intervals, or to titrate the separate compositions with respect to each other. To facilitate compliance, the kit typically includes administration instructions. In combination therapies, the compound of the invention and the other therapeutic agent can be manufactured and/or formulated by the same manufacturer or by different manufacturers. Furthermore, the compound of the invention and the other therapeutic agent can be combined in a combination therapy: (i) prior to delivery of the combination product to physicians (for example, in the case of a kit comprising the compound of the invention and the other therapeutic agent); (ii) by the physician himself (or under the guidance of the physician) shortly before administration; (iii) in the patient himself, for example, during sequential administration of the compound of the invention and the other therapeutic agent. Accordingly, described herein is a method of treating a disease or condition mediated by CDK4/6 inhibition, wherein the medicament is prepared for administration with another therapeutic agent. Also described herein is a method of treating a disease or condition mediated by CDK4/6 inhibition, wherein the medicament is administered with a compound of the present invention. Also described herein is a method of treating a disease or condition mediated by CDK4/6 inhibition, wherein the compound of the invention, or a pharmaceutically acceptable salt thereof, is prepared for administration with another therapeutic agent. Also described herein is another therapeutic agent for use in a method of treating a disease or condition mediated by CDK4/6 inhibition, wherein the other therapeutic agent is prepared for administration with a compound of the invention, or a pharmaceutically acceptable salt thereof. Also described herein is a compound of the invention, or a pharmaceutically acceptable salt thereof, for use in a method of treating a disease or condition mediated by CDK4/6 inhibition, wherein the compound of the invention, or a pharmaceutically acceptable salt thereof, is administered with another therapeutic agent. Also described herein is another therapeutic agent for use in a method of treating a disease or condition mediated by CDK4/6 inhibition, wherein the other therapeutic agent is administered with a compound of the invention, or a pharmaceutically acceptable salt thereof. Also described herein is the use of a compound of the invention, or a pharmaceutically acceptable salt thereof, for treating a disease or condition mediated by CDK4/6, wherein the patient has been previously treated (e.g., within 24 hours) with another therapeutic agent. Also described herein is the use of another therapeutic agent to treat a CDK4/6 mediated disease or condition, wherein the patient has been previously treated (e.g., within 24 hours) with a compound of the invention, or a pharmaceutically acceptable salt thereof.

The other therapeutic agent is selected from an anti-inflammatory, antiproliferative, chemotherapeutic, immunosuppressive, anticancer, cytotoxic agent or kinase inhibitor other than a compound of the present invention, or a salt thereof. Additional examples of agents that can be administered in combination with the compounds of the invention include, but are not limited to, a PTK inhibitor, cyclosporine A, CTLA4-Ig, antibodies selected from anti-iCAM-3, anti-IL-2 receptor, anti-CD45RB, anti-CD2, anti-CD3, anti-CD4, anti-CD80, anti-CD86 and OKT3 monoclonal antibody, agents that block the interaction between CD40 and gp39, fusion proteins constructed from CD40 and gp39, inhibitors of NF-kappa B function, nonsteroidal anti-inflammatory drugs, steroids, gold compounds, antiproliferative agents, FK506, mycophenolate mofetil, cytotoxic drugs, TNF-α inhibitors, anti-TNF antibodies or soluble TNF receptor, rapamycin, mTOR inhibitors, leflunimide, cyclooxygenase-2, paclitaxel, cisplatin, carboplatin, doxorubicin, carminomycin, daunorubicin, aminopterin, methotrexate, methopterin, mitomycin C, ecteinascidin 743, porfiromycin, 5-fluorouracil, 6-mercaptopurine, gemcitabine, cytosine arabinoside, podophyllotoxin, etoposide, etoposide phosphate, teniposide, melphalan, vinblastine, vincristine, leurosidine, epothilone, vindesine, leurosine, B-Raf inhibitor, MEK inhibitor, PI3K inhibitor, HSP90 inhibitor, CDK1 inhibitor, CDK2 inhibitor, CDK5 inhibitor, CDK7 inhibitor, CDK8 inhibitor, CDK9 inhibitor, EGFR inhibitor, FGFR inhibitor, PDGFR, Her2neu inhibitor, FLT3 inhibitor, Androgen, glucocorticoid and prosterone receptor antagonists, SO inhibitor, WNT inhibitor, Bel inhibitor, IAP inhibitor, CL inhibitor, MD 2 inhibitor, p52 inhibitor, proteasome inhibitors (Velcade) or derivatives thereof.

Specific individual combinations that may provide particular treatment benefits include co-treatment of patients with mantle cell lymphoma or pancreatic cancer with mTOR inhibitors, such as everolimus.

A compound of the present invention may also be used in combination with other agents, e.g., an additional protein kinase inhibitor that may or may not be a compound of the invention, for the treatment of a protein kinase-associated disorder in a subject. By the term “combination” is meant a fixed combination in unit dosage form or a kit of parts for combined administration wherein a compound of the present invention and a combination partner may be administered independently at the same time or separately within time intervals that specifically allow the combination partners to exhibit a cooperative, e.g., synergistic effect, or any combination thereof.

The compounds of the invention may be administered, simultaneously or sequentially, with an anti-inflammatory, antiproliferative, chemotherapeutic, immunosuppressive, anticancer, cytotoxic or kinase inhibitor agent other than a compound of Formula I or a pharmaceutically acceptable salt thereof. Additional examples of agents that can be administered in combination with the compounds of the invention include, but are not limited to, a PTK inhibitor, cyclosporine A, CTLA4-Ig, antibodies selected from anti-ICAM-3, anti-IL-2 receptor, anti-CD45RB, anti-CD2, anti-CD3, anti-CD4, anti-CD80, anti-CD86 and OKT3 monoclonal antibody, agents that block the interaction between CD40 and gp39, fusion proteins constructed from CD40 and gp39, inhibitors of NF-kappa B function, nonsteroidal anti-inflammatory drugs, steroids, gold compounds, antiproliferative agents, FK506, mycophenolate mofetil, cytotoxic drugs, TNF-ct inhibitors, anti-TNF antibodies or soluble TNF receptor, rapamycin, leflunimide, cyclooxygenase-2 inhibitors, paclitaxel, cisplatin, carboplatin, doxorubicin, carminomycin, daunorubicin, aminopterin, methotrexate, methopterin, mitomycin C, ecteinascidin 743, porfiromycin, 5-fluorouracil, 6-mercaptopurine, gemcitabine, cytosine arabinoside, podophyllotoxin, etoposide, etoposide phosphate, teniposide, melphalan, vinblastine, vincristine, leurosidine, epothilone, vindesine, leurosine or derivatives thereof.

A compound of the invention and any additional agent can be formulated in separate dosage forms. Alternatively, to reduce the number of dosage forms administered to a patient, the compound of the invention and any additional agent can be formulated together in any combination. For example, the inhibitory compound of the invention can be formulated in one dosage form, and the additional agent can be formulated together in another dosage form. Any separate dosage forms can be administered at the same time or at different times.

Alternatively, a composition of this invention may comprise an additional agent as described herein. Each component may be present in individual compositions, combined compositions, or a single composition.

Pharmaceutical compositions and their administration

The compositions and methods disclosed herein can be used to treat an individual in need thereof. In certain embodiments, the individual is a mammal, such as a human, or a non-human mammal. When administered to an animal, such as a human, the composition or compound is preferably administered as a pharmaceutical composition comprising, for example, a disclosed compound and a pharmaceutically acceptable carrier.

Pharmaceutically acceptable carriers are well known in the art and include, for example, aqueous solutions such as water or physiologically buffered saline, or other solvents or vehicles such as glycols, glycerol, oils such as olive oil, or injectable organic esters. In preferred embodiments, when such pharmaceutical compositions are for human administration, particularly for invasive routes of administration (i.e., routes, such as injection or implantation, that prevent transport or diffusion across an epithelial barrier), the aqueous solution is pyrogen-free or substantially pyrogen-free. Excipients may be chosen, for example, to effect delayed release of an agent or to selectively target one or more cells, tissues, or organs. The pharmaceutical composition may be in dosage unit form, such as a tablet, capsule (including sprinkle capsule and gelatin capsule), granule, lyophilizate for reconstitution, powder, solution, syrup, suppository, injection, or the like. The composition may also be present in a transdermal delivery system, for example, a skin patch. The composition may also be present in a solution suitable for topical administration, such as an ointment or cream.

A pharmaceutically acceptable carrier may contain physiologically acceptable agents that act, for example, to stabilize, increase the solubility, or increase the absorption of a compound such as a compound of the invention. Such physiologically acceptable agents include, for example, carbohydrates, such as glucose, sucrose, or dextrans, antioxidants, such as ascorbic acid or glutathione, chelating agents, low molecular weight proteins, or other stabilizers or excipients. The choice of a pharmaceutically acceptable carrier, which includes a physiologically acceptable agent, depends, for example, on the route of administration of the composition. The preparation of the pharmaceutical composition may be a self-emulsifying drug delivery system or a self-microemulsifying drug delivery system. The pharmaceutical composition (preparation) may also be a liposome or other polymeric matrix, which may have incorporated therein, for example, a compound of the invention. Liposomes, for example, which comprise phospholipids or other lipids, are non-toxic, physiologically acceptable, and metabolizable carriers that are relatively simple to produce and administer.

The phrase “pharmaceutically acceptable” is used herein to refer to those compounds, materials, compositions and/or dosage forms that, within the scope of sound medical judgment, are suitable for use in contact with human and animal tissues without undue toxicity, irritation, allergic response or other problem or complication, commensurate with a reasonable benefit/risk ratio.

The phrase “pharmaceutically acceptable carrier” as used herein means a pharmaceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, solvent, or encapsulating material. Each carrier must be “acceptable” in the sense of being compatible with the other ingredients of the formulation and not deleterious to the patient. Some examples of materials that may serve as pharmaceutically acceptable carriers include: (1) sugars, such as lactose, glucose, and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose and its derivatives, such as sodium carboxymethylcellulose, ethylcellulose, and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol, and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) phosphate buffer solutions; and (21) other compatible nontoxic substances employed in pharmaceutical formulations.

A pharmaceutical composition (preparation) can be administered to a subject by any of several routes of administration, including, for example, orally (e.g., aqueous or non-aqueous solutions or suspensions, tablets, capsules (including sprinkle capsules and gelatin capsules), boluses, powders, granules, pastes for application to the tongue); absorption through the oral mucosa (e.g., sublingually); anally, rectally, or vaginally (e.g., as a pessary, cream, or foam); parenterally (including intramuscularly, intravenously, subcutaneously, or intrathecally, such as a sterile solution or suspension); nasally; intraperitoneally; subcutaneously; transdermally (e.g., as a patch applied to the skin); and topically (e.g., as a cream, ointment, or spray applied to the skin, or as an eye drop). The compound can also be formulated for inhalation. In certain embodiments, a compound may simply be dissolved or suspended in sterile water. Details of appropriate routes of administration and suitable compositions therefor can be found, for example, in U.S. Patent Nos. 6,110,973, 5,763,493, 5,731,000, 5,541,231, 5,427,798, 5,358,970, and 4,172,896, as well as the patents cited therein.

The formulations may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy. The amount of active ingredient that may be combined with a carrier material to produce a single dosage form will vary depending on the host being treated and the particular mode of administration. The amount of active ingredient that may be combined with a carrier material to produce a single dosage form will generally be that amount of the compound that produces a therapeutic effect. Generally, of one hundred percent, this amount will range from about 1 percent to about ninety-nine percent of the active ingredient, preferably from about 5 percent to about 70 percent, more preferably from about 10 percent to about 30 percent.

Methods for preparing these formulations or compositions include the step of bringing into association an active compound, such as a compound of the invention, with the carrier and, optionally, one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association a compound of the present invention with liquid carriers or finely divided solid carriers, or both, and then, if necessary, forming the product.

Formulations of the invention suitable for oral administration may be in the form of capsules (including sprinkle capsules and gelatin capsules), cachets, pills, tablets, lozenges (using a flavored base, usually sucrose and acacia or tragacanth), lyophilizate, powders, granules, or as a solution or suspension in an aqueous or non-aqueous liquid, or as an oil-in-water or water-in-oil liquid emulsion, or as an elixir or syrup, or as lozenges (using an inert base, such as gelatin and glycerin, or sucrose and acacia) and/or as mouthwashes and the like, each containing a predetermined amount of a compound of the present invention as an active ingredient. The compositions or compounds may also be administered in the form of a bolus, electuary, or paste.

In preparing solid dosage forms for oral administration (capsules (including sprinkle capsules and gelatin capsules), tablets, pills, dragees, powders, granules and the like), the active ingredient is mixed with one or more pharmaceutically acceptable carriers, such as sodium citrate or dicalcium phosphate, and/or any of the following: (1) fillers or extenders, such as starches, lactose, sucrose, glucose, mannitol and/or silicic acid; (2) binders, such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose and/or acacia; (3) humectants, such as glycerol; (4) disintegrating agents, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates and sodium carbonate; (5) solution retarding agents, such as paraffin; (6) absorption accelerators, such as quaternary ammonium compounds; (7) wetting agents, such as, for example, cetyl alcohol and glycerol monostearate; (8) absorbents, such as kaolin and bentonite clay; (9) lubricants, such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof; (10) complexing agents, such as modified and unmodified cyclodextrins; and (11) coloring agents. In the case of capsules (including sprinkle capsules and gelatin capsules), tablets, and pills, the pharmaceutical compositions may also comprise buffering agents. Solid compositions of a similar type may also be employed as fillers in soft and hard gelatin capsules using excipients such as lactose or milk sugars, as well as high molecular weight polyethylene glycols and the like.

A tablet may be manufactured by compression or molding, optionally with one or more accessory ingredients. Compressible tablets may be prepared using a binder (e.g., gelatin or hydroxypropyl methylcellulose), lubricant, inert diluent, preservative, disintegrant (e.g., sodium starch glycolate or cross-linked sodium carboxymethylcellulose), surface active agent, or dispersant. Molded tablets may be manufactured by molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent.

Tablets and other solid dosage forms of the pharmaceutical compositions, such as dragees, capsules (including sprinkle capsules and gelatin capsules), pills, and granules, may optionally be scored or prepared with coatings and shells, such as enteric coatings and other coatings well known in the pharmaceutical formulation art. They may also be formulated to provide slow or controlled release of the active ingredient they contain using, for example, hydroxypropyl methylcellulose in varying proportions to provide the desired release profile, other polymeric matrices, liposomes, and/or microspheres. They may be sterilized, for example, by filtration through a bacteria-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions that can be dissolved in sterile water or some other sterile injectable medium immediately prior to use. These compositions may also optionally contain opacifying agents and may be of a composition that releases the active ingredient(s) only, or preferentially, in a certain portion of the gastrointestinal tract, optionally in a delayed manner. Examples of embedding compositions that may be used include polymeric substances and waxes. The active ingredient may also be presented in microencapsulated form, if appropriate, with one or more of the excipients described above.

Liquid dosage forms useful for oral administration include pharmaceutically acceptable emulsions, lyophilizates for reconstitution, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the active ingredient, the liquid dosage forms may contain inert diluents commonly used in the art, such as, for example, water or other solvents, cyclodextrins and derivatives thereof, solubilizing and emulsifying agents, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils (in particular, cottonseed, peanut, corn, germ, olive, castor and sesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene glycols and sorbitan fatty acid esters, and mixtures thereof.

In addition to inert diluents, oral compositions may also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening agents, flavoring agents, coloring agents, perfuming agents, and preservatives.

Suspensions, in addition to the active compounds, may contain suspending agents such as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, and mixtures thereof.

Formulations of pharmaceutical compositions for rectal, vaginal or urethral administration may be presented as a suppository, which may be prepared by mixing one or more active compounds with one or more suitable non-irritating excipients or carriers comprising, for example, cocoa butter, polyethylene glycol, a suppository wax or a salicylate, and which is solid at room temperature, but liquid at body temperature and will therefore melt in the rectum or vaginal cavity and release the active compound.

Formulations of pharmaceutical compositions for oral administration may be presented as a mouthwash, or an oral spray, or an oral ointment.

Alternatively or additionally, compositions can be formulated for delivery via a catheter, stent, wire, or other intraluminal device. Delivery via such devices can be particularly useful in cases of delivery to the bladder, urethra, ureter, rectum, or intestine.

Formulations that are suitable for vaginal administration also include pessaries, tampons, creams, gels, pastes, foams or spray formulations containing carriers known in the art to be appropriate.

Dosage forms for topical or transdermal administration include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches, and inhalants. The active compound can be mixed under sterile conditions with a pharmaceutically acceptable carrier and any preservatives, buffers, or propellants that may be required.

Ointments, pastes, creams and gels may contain, in addition to an active compound, excipients such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide, or mixtures thereof.

Powders and aerosols may contain, in addition to an active ingredient, excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicates, and polyamide powder, or mixtures of these substances. Aerosols may also contain common propellants, such as chlorofluorohydrocarbons and unsubstituted volatile hydrocarbons, such as butane and propane.

Transdermal patches have the additional advantage of providing controlled delivery of a compound of the present invention to the body. Such dosage forms can be prepared by dissolving or dispersing the active compound in a suitable medium. Absorption enhancers can also be used to increase the flux of the compound through the skin. The rate of such flux can be controlled by providing a rate-controlling membrane or by dispersing the compound in a polymer matrix or gel.

Ophthalmic formulations, eye ointments, powders, solutions, and the like are also contemplated within the scope of this invention. Exemplary ophthalmic formulations are described in U.S. Publication Nos. 2005/0080056, 2005/0059744, 2005/0031697, and 2005/004074; and U.S. Patent No.

6,583,124, the contents of which are incorporated herein by reference. If desired, liquid ophthalmic formulations have properties similar to those of tear fluids, aqueous humor, or vitreous humor, or are compatible with such fluids.

The phrases “parenteral administration” and “administered parenterally” as used herein mean modes of administration other than enteral and topical administration, usually by injection, and include, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, and intrasternal injection and infusion.

Pharmaceutical compositions suitable for parenteral administration comprise one or more active compounds in combination with one or more pharmaceutically acceptable sterile isotonic aqueous or non-aqueous solutions, dispersions, suspensions or emulsions or sterile powders which can be reconstituted into sterile injectable solutions or dispersions just prior to use, which may contain antioxidants, buffers, bacteriostats, solutes which render the formulation isotonic with the blood of the intended recipient or suspending or thickening agents.

Examples of suitable aqueous and non-aqueous carriers which may be employed in the pharmaceutical compositions of the invention include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol and the like) and suitable mixtures thereof, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Adequate fluidity may be maintained, for example, by the use of coating materials such as lecithin, by maintaining the required particle size in the case of dispersions and by the use of surfactants.

These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents, and dispersing agents. Prevention of the action of microorganisms can be ensured by the inclusion of various antibacterial and antifungal agents, for example, parabens, chlorobutanol, sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like, in the compositions. Furthermore, prolonged absorption of the injectable pharmaceutical form can be achieved by the inclusion of agents that delay absorption, such as aluminum monostearate and gelatin.

In some cases, to prolong the effect of a drug, it is desirable to delay drug absorption through subcutaneous or intramuscular injection. This can be achieved by using a liquid suspension of crystalline or amorphous material with low water solubility. The rate of drug absorption then depends on its dissolution rate, which, in turn, may depend on the crystalline size and shape. Alternatively, delayed absorption of a parenterally administered drug form is achieved by dissolving or suspending the drug in an oily vehicle.

Injectable depot forms are made by forming microencapsulated matrices of the compounds in question in biodegradable polymers such as polylactide-polyglycolide. Depending on the ratio of drug to polymer, and the nature of the particular polymer used, the rate of drug release can be controlled. Examples of biodegradable polymers include poly(orthoesters) and poly(anhydrides). Depot injectable formulations are also prepared by entrapping the drug in liposomes or microemulsions that are compatible with body tissue.

For use in the methods described herein, the active compounds may be provided per se or as a pharmaceutical composition containing, for example, between 0.1% and about 99.5% (more preferably, between 0.5% and about 90.0%) of active ingredient in combination with a pharmaceutically acceptable carrier.

While specific embodiments of the subject invention have been disclosed, the foregoing specification is illustrative and not restrictive. Many variations of the invention will become apparent to those skilled in the art upon review of this specification and the claims that follow. The full scope of the invention should be determined by reference to the claims and specification.

Exemplification

Synthetic protocols

Table 1.

Example 1: Synthesis of compounds A1, A2, A3, A5, A6, A7, A8, A9, A10, A11, A12, A13, A14, A15, A16, A17, A18, A19, A20, A21, A22, A29, A45, A47, A49, A50, A52, A53, A54, A55, A56, A57, A58, A59, A60

To a cooled (0 °C) solution of allyl 4-benzoyl-3-oxomorpholine-2-carboxylate (17.5 g, 60.5 mmol) in DMF (60 mL) was added iodomethane (7.5 mL, 121 mmol), followed by cesium carbonate (29.6 g, 90.8 mmol). The reaction was stirred for 2 h, at which time saturated ammonium chloride (aq., 300 mL) and water (200 mL) were added. The mixture was extracted with Et2O (3 x 300 mL), and the combined organic layers were dried over MgSO4 and concentrated in vacuo. The crude compound was purified using flash chromatography on SiO2 (gradient elution of hexanes with EtOAc = 0-50%) to afford allyl 4-benzoyl-2-methyl-3-oxomorpholine-2-carboxylate (14.6 g, 80% yield) as a white solid.

MS: [M+H]+ m/z 304.1.

Pd2(dba)3 (440 mg, 0.48 mmol), (S)-(p-CF3)3-t-BuPHOX (710 mg, 1.2 mmol), and MTBE (100 mL) were added to a Schlenk flask equipped with a stir bar. The mixture was stirred for 30 min, after which allyl 4-benzoyl-2-methyl-3-oxomorpholine-2-carboxylate (14.6 g, 48 mmol) was added as a solution in MTBE (220 mL). The flask was sealed and heated at 50 °C for two days. Once complete, the reaction was cooled to room temperature and vented to release the evolved CO2. The mixture was filtered through Celite, washed with EtOAc, and concentrated in vacuo. The crude compound was purified using flash chromatography on SiO2 (gradient elution of hexanes with EtOAc = 0-20%) to provide (S)-2-allyl-4-benzoyl-2-methylmorpholin-3-one (12.1 g, 98% yield, 98% ee) as a white solid.

MS: [M+H]+ m/z 260.1.

Potassium carbonate (647 mg, 4.7 mmol) was added to a mixture of (S)-2-allyl-4-benzoyl-2-methylmorpholin-3-one (12.1 g, 47 mmol) in THF/MeOH (2:1, 24 mL). The flask was fitted with a reflux condenser, and the reaction was heated under reflux for 3 h. Upon completion, the mixture was cooled, filtered through Celite, washed with CHCl, and concentrated in vacuo. The crude compound was purified using flash chromatography on SO (gradient elution of CHCl with 2% NH/MeOH = 0–20%) to afford (S)-2-allyl-2-methylmorpholin-3-one (6.45 g, 89% yield) as a colorless oil.

MS: [M+H]+ m/z 156.1.

To a cooled (0 °C) solution of (S)-2-allyl-2-methylmorpholin-3-one (6.45 g, 42 mmol) in THF (210 mL) was added lithium aluminum hydride (4.73 g, 125 mmol). The mixture was warmed to room temperature and heated at 60 °C for 3 h. Upon completion, the mixture was cooled to 0 °C and Et2O (300 mL) was added. Water (5.7 mL) was slowly added, followed by sodium hydroxide solution (1 M aq., 5.7 mL), and the mixture was warmed to room temperature and stirred for 15 min. The mixture was dried with MgSO4, filtered through Celite, and concentrated in vacuo to afford (S)-2-allyl-2-methylmorpholine (5.29 g) as a colorless oil. The compound was used immediately without further purification.

Mass [M H] 142.1.

Compound A45

To a solution of (S)-2-allyl-2-methylmorpholine (5.29 g, 38 mmol) and 6-((5-fluoro-4-(4-fluoro-1-isopropyl-2-methylMH-benzo[d]imidazol-6-yl)pyrimidin-2-yl)amino)nicotinaldehyde (12.75 g, 31 mmol) in DMSO (315 mL) was added sodium triacetoxyborohydride (13.23 g, 62 mmol), followed by acetic acid (18 mL, 312 mmol). The reaction was heated to 60 °C and stirred for 24 h. Upon completion, the mixture was cooled to 0 °C, and aqueous sodium hydroxide solution (1 M, 630 mL) was added, followed by water (200 mL). The resulting heterogeneous mixture was stirred for 1 h, then filtered to collect the solid, rinsed with cold water and finally lyophilized to yield (S)-N-(5-((2-allyl-2-methylmorpholino)methyl)pyridin-2-yl)-5-fluoro-4-(4-fluoro-1-isopropyl-2-methyl-1H-benzo[d]imidazol-6-yl)pyrimidin-2-amine (15.82 g) as a gray solid. The compound was used without further purification.

MS: [M+H]+ m/z 534.3.

Compound B1

To a solution of (S)-N-(5-((2-allyl-2-methylmorpholino)methyl)pyridin-2-yl)-5-fluoro-4-(4-fluoro-1-isopropyl-2-methyl-1H-benzo[d]imidazol-6-yl)pyrimidin-2-amine (15.82 g, 30 mmol) in THF/H2O (2:1, 750 mL) was added osmium tetroxide (4% in H2O, 9.4 mL, 1.48 mmol), followed by sodium periodate (19.02 g, 90 mmol). The reaction was sonicated for 2.5 h and then stirred for 4.5 h. Upon completion, saturated sodium thiosulfate (aq, 600 mL) was added, followed by sodium hydroxide solution (1 M aq, 400 mL), and the mixture was extracted with CHCl (3 x 800 mL). The organic layers were combined and dried over Na2SO4, and concentrated in vacuo to afford (S)-2-(4-((6-((5-fluoro-4-(4-fluoro-1-isopropyl-2-methyl-1H-benzo[d]imidazol-6-yl)pyrimidin-2-yl)amino)pyridin-3-yl)methyl)-2-methylmorpholin-2-yl)acetaldehyde (15.94 g) as a brown solid. The compound was used without further purification.

MS: [M+H]+ m/z 536.3.

Compound A1

To a cooled (0 °C) solution of (S)-2-(4-((6-((5-fluoro-4-(4-fluoro-1-isopropyl-2-methyl-1H-benzo[d]imidazol-6-yl)pyrimidin-2-yl)amino)pyridin-3-yl)methyl)-2-methylmorpholin-2-yl)acetaldehyde (15.94 g, 30 mmol) in CHCh/MeOH (2:1, 300 mL) was added dimethylamine (2 M in THF, 75 mL, 150 mmol). After 15 min of stirring, sodium triacetoxyborohydride (12.63 g, 60 mmol) was added and the reaction was warmed to room temperature and stirred for 2 h. Upon completion, sodium hydroxide solution (0.5 M aq., 600 mL) was added and the mixture was extracted with CHCl (3 x 500 mL). The organic layers were combined and dried over NaSO, and concentrated in vacuo. The crude compound was purified using flash chromatography on SO (gradient elution of CHCl with 2% NHMeOH = 0-40%) to afford (S)-N-(5-((2-(2-(dimethylamino)ethyl)-2-methylmorpholino)methyl)pyridin-2-yl)-5-fluoro-4-(4-fluoro-1-isopropyl-2-methyl-1H-benzo[d]imidazol-6-yl)pyrimidin-2-amine as a light orange oil. The oil was dissolved in water containing methanesulfonic acid (1.05 equiv.) and the solution was lyophilized to yield 12.6 g of the mesylate salt as a tan solid.

Mesylate salt spectra

1H NMR (400 MHz, DMSO-da) 58.70(d, J = 3.8 Hz, 1H), 8.30 (d, J = 1.3 Hz, 1H), 8.24 (dd, J = 8.5, 0.8 Hz, 1H), 8.22 - 8.18 (m, 1H), 7.75 - 7.64 (m, 2H), 4.85 (hept, J = 7.0 Hz, 1H), 3.59 (t, J = 4.8 Hz, 2H), 2.64 (s, 3H), 2.42 - 2.01 (m, 17H), 1.87 - 1.76 (m, 1H), 1.63 (d, J = 6.9 Hz, 6H), 1.53 (ddd, J = 13.2, 10.4, 5.9 Hz, 1H), 1.11 (s, 3H). MS: [M+H]+ m/z 565.3.

Compound B2

(R)-2-(4-((6-((5-fluoro-4-(4-fluoro-1-isopropyl-2-methyl-1H-benzo[d]imidazol-6-yl)pyrimidin-2-yl)amino)pyridin-3-yl)methyl)-2-methylmorpholin-2-yl)acetaldehyde was prepared in the same manner as compound B1 (above) using (R)-(p-CF3)3-t-BuPHOX as a chiral catalyst. MS: [M+H]+ m/z 536.3.

Compound A2

(S)-N-(5-((2-(2-(dimethylamino)ethyl)-2-methylmorpholino)methyl)pyridin-2-yl)-5-fluoro-4-(4-fluoro-1-isopropyl-2-methyl-1H-benzo[d]imidazol-6-yl)pyrimidin-2-amine and its mesylate salt of compound B1 in the same manner as described for compound A1 (above).

Compound A5

To a cooled (0 °C) solution of (R)-2-(4-((6-((5-fluoro-4-(4-fluoro-1-isopropyl-2-methyl-1H-benzo[d]imidazol-6-yl)pyrimidin-2-yl)amino)pyridin-3-yl)methyl)-2-methylmorpholin-2-yl)acetaldehyde (90 mg, 0.17 mmol) in CHCl3 (2.1 mL) was added MgSO4 (120 mg) followed by methylamine (2 M in MeOH, 0.84 mL, 1.7 mmol). After warming to room temperature and stirring for 20 h, sodium borohydride (13 mg, 0.34 mmol) was added and the reaction was stirred for 4 h. Upon completion, water (6 mL) and brine (2 mL) were added and the mixture was extracted with CHCl (3 x 4 mL). The organic layers were combined and dried over NaSO, and concentrated in vacuo. The crude compound was purified using flash chromatography on SiO (gradient elution of CHCl with 2% NHMeOH = 0-40%) to afford (R)-5-fluoro-4-(4-fluoro-1-isopropyl-2-methyl-1H-benzo[d]imidazol-6-yl)-N-(5-((2-methyl-2-(2-(methylamino)ethyl)morpholino)methyl)pyridin-2-yl)pyrimidin-2-amine as a light orange oil. The oil was dissolved in water containing methanesulfonic acid (1.05 equiv.) and the solution was lyophilized to yield 28.3 mg of the mesylate salt as a tan solid.

Mesylate salt spectra

1H NMR (400 MHz, DMSO-da) 58.70(d, J = 3.8 Hz, 1H), 8.29 (d, J = 1.3 Hz, 1H), 8.26 - 8.20 (m, 2H), 7.72 -7.67 (m, 2H), 4.85 (hept, J = 6.8 Hz, 1H), 3.61 (t, J = 4.9 Hz, 2H), 2.64 (s, 3H), 2.60 - 2.53 (m, 4H), 2.37 - 2.27 (m, 9H), 2.20 (d, J = 11.1 Hz, 1H), 2.11 (d, J = 11.1 Hz, 1H), 2.05 - 1.91 (m, 1H), 1.63 (d, J = 7.0 Hz, 6H), 1.58 - 1.45 (m, 1H), 1.12 (s, 3H).

MS: [M+H]+ m/z 551.3.

Compound A22

(S)-5-fluoro-4-(4-fluoro-1-isopropyl-2-methyl-1H-benzo[d]imidazol-6-yl)-N-(5-((2-methyl-2-(2-(methylamino)ethyl)morpholino)methyl)pyridin-2-yl)pyrimidin-2-amine and its mesylate salt of compound B1 in the same manner as described for compound A5 (above).

Compound A49

To a cooled (0 °C) solution of (R)-2-(4-((6-((5-fluoro-4-(4-fluoro-1-isopropyl-2-methyl-1H-benzo[d]imidazol-6-yl)pyrimidin-2-yl)amino)pyridin-3-yl)methyl)-2-methylmorpholin-2-yl)acetaldehyde (1.37 g, 2.13 mmol) in CHCh/MeOH (2:1, 30 mL) was added sodium borohydride (121 mg, 3.2 mmol) and the reaction was stirred for 2 h. After completion, aqueous hydrochloric acid (1 M, 20 mL) was added and the mixture was stirred for an additional 15 min. Sodium hydroxide solution (0.5 M, 200 mL) was added and the mixture was extracted with CHCl (3 x 150 mL). The organic layers were combined and dried over NaSO, and concentrated in vacuo. The crude compound was purified using flash chromatography on C18 reversed-phase SiO (gradient elution of H2O 0.1% AcOH with MeCN = 5-50%) to afford (R)-2-(4-((6-((5-fluoro-4-(4-fluoro-1-isopropyl-2-methyl-1H-benzo[d]imidazol-6-yl)pyrimidin-2-yl)amino)pyridin-3-yl)methyl)-2-methylmorpholin-2-yl)ethan-1-ol as a light orange oil. The oil was dissolved in water containing methanesulfonic acid (1.05 equiv.) and the solution was lyophilized to yield 400 mg of the mesylate salt as a tan solid.

Mesylate salt spectra

1H NMR (400 MHz, DMSO-d6) 58.70 (d, J = 2.8 Hz, 1H), 8.33 - 8.17 (m, 3H), 7.75 - 7.64 (m, 2H), 4.84 (p, J = 7.0 Hz, 1H), 3.65 - 3.55 (m, 2H), 3.49 - 3.39 (m, 2H), 2.65 (s, 3H), 2.39 - 2.25 (m, 6H), 2.24 - 2.07 (m, 2H), 1.97 - 1.83 (m, 1H), 1.69 - 1.52 (m, 8H), 1.14 (s, 3H). MS: [M+H]+ m/z 538.2.

Compound A50

(S)-2-(4-((6-((5-fluoro-4-(4-fluoro-1-isopropyl-2-methyl-1H-benzo[d]imidazol-6-yl)pyrimidin-2-yl)amino)pyridin-3-yl)methyl)-2-methylmorpholin-2-yl)ethan-1-ol and its mesylate salt of compound B1 in the same manner as described for compound A49 (above).

Compound A19

To a cooled (0 °C) solution of (R)-2-(4-((6-((5-fluoro-4-(4-fluoro-1-isopropyl-2-methyl-1H-benzo[d]imidazol-6-yl)pyrimidin-2-yl)amino)pyridin-3-yl)methyl)-2-methylmorpholin-2-yl)acetaldehyde (30 mg, 0.056 mmol) in CHCl3/MeOH (2:1, 1 mL) was added morpholine (50<j>L, 0.56 mmol). After 15 min of stirring, sodium triacetoxyborohydride (24 mg, 0.1 L mmol) was added and the reaction was warmed to room temperature and stirred for 2 h. Upon completion, sodium hydroxide solution (0.5 M aq., 3 mL) was added and the mixture was extracted with CHCl (3 x 2 mL). The organic layers were combined and dried over NaSO, and concentrated in vacuo. The crude compound was purified using flash chromatography on SiO (gradient elution of CHCl with 2% NHMeOH = 0-40%) to afford (R)-5-fluoro-4-(4-fluoro-1-isopropyl-2-methyl-1H-benzo[d]imidazol-6-yl)-N-(5-((2-methyl-2-(2-morpholinoethyl)morpholino)methyl)pyridin-2-yl)pyrimidin-2-amine as a light orange oil. The oil was dissolved in water containing methanesulfonic acid (1.05 equiv.) and the solution was lyophilized to yield 17.8 mg of the mesylate salt as a tan solid.

Mesylate salt spectra

1H NMR (400 MHz, DMSO-da) 88.70 (d, J = 3.8 Hz, 1H), 8.30 (d, J = 1.3 Hz, 1H), 8.28 - 8.19 (m, 2H), 7.75 -7.65 (m, 2H), 4.85 (hept, J = 6.9 Hz, 1H), 3.60 (t, J = 4.8 Hz, 2H), 3.52 (t, J = 4.7 Hz, 4H), 2.65 (s, 3H), 2.44 -2.17 (m, 13H), 2.10 (d, J = 11.1 Hz, 1H), 1.85 (q, J = 13.3, 10.7 Hz, 1H), 1.71 - 1.51 (m, 8H), 1.12 (s, 3H). MS:

[M+H]+ m/z 607.3.

Compound A7

To a cooled (0 °C) solution of (R)-2-(4-((6-((5-fluoro-4-(4-fluoro-1-isopropyl-2-methyl-1H-benzo[d]imidazol-6-yl)pyrimidin-2-yl)amino)pyridin-3-yl)methyl)-2-methylmorpholin-2-yl)acetaldehyde (30 mg, 0.056 mmol) in CHCh/MeOH (2:1, 1 mL) was added MgSO4 (40 mg) followed by t-butylamine (60 L, 0.56 mmol). After warming to room temperature and stirring for 18 h, sodium borohydride (5 mg, 0.11 mmol) was added and the reaction was stirred for 4 h. After completion, water (3 mL) and brine (1 mL) were added and the mixture was extracted with CHCl (3 x 2 mL). The organic layers were combined and dried over NaSO, and concentrated in vacuo. The crude compound was purified using flash chromatography on SiO (gradient elution of CHCl with 2% NHMeOH = 0-40%) to afford (R)-N-(5-((2-(2-(tert-butylamino)ethyl)-2-methylmorpholino)methyl)pyridin-2-yl)-5-fluoro-4-(4-fluoro-1-isopropyl-2-methyl-1H-benzo[d]imidazol-6-yl)pyrimidin-2-amine as a light orange oil. The oil was dissolved in water containing methanesulfonic acid (1.05 equiv.) and the solution was lyophilized to yield 14.2 mg of the mesylate salt as a tan solid.

Mesylate salt spectra

1H NMR (400 MHz, DMSO-da) 88.70 (d, J = 3.8 Hz, 1H), 8.29 (d, J = 1.4 Hz, 1H), 8.27 - 8.20 (m, 2H), 7.73 -7.66 (m, 2H), 4.84 (p, J = 6.9 Hz, 1H), 3.64 -3.59 (m, 2H), 2.64 (s, 3H), 2.60 -2.53 (m, 2H), 2.43 -2.28 (m, 7H), 2.23 (d, J = 11.1 Hz, 1H), 2.10 (d, J = 11.1 Hz, 1H), 1.95 (d, J = 8.3Hz, 1H), 1.63 (dd, J = 6.9, 1.3 Hz, 6H), 1.59 - 1.42 (m, 1H), 1.14 (s, 3H), 1.07 (s, 9H). MS: [M+H]+ m/z 593.3.

Compound A8

To a solution of (R)-5-fluoro-4-(4-fluoro-1-isopropyl-2-methyl-1H-benzo[d]imidazol-6-yl)-N-(5-((2-methyl-2-(2-(methylamino)ethyl)morpholino)methyl)pyridin-2-yl)pyrimidin-2-amine (17 mg, 0.031 mmol) in CHCl3 was added acetyl chloride (4.5 pL, 0.062 mmol) followed by triethylamine (20 pL, 0.12 mmol). After stirring for 2 h, sodium hydroxide solution (1 M aq., 3 mL) was added and the mixture was stirred for 1 h. Upon completion, water (3 mL) was added and the mixture was extracted with CHCl (3 x 1.5 mL). The organic layers were combined and dried over Na2SO4, and concentrated in vacuo. The crude compound was stirred with lithium hydroxide solution (1 M aq., 2 mL) in DMF/MeOH (2 mL) for 2 h, before water (3 mL) was added and the mixture was extracted with CHCl3 (3 x 1.5 mL). The organic layers were combined and dried over Na2SO4, and concentrated in vacuo. The crude compound was purified using flash chromatography on SO2 (gradient elution of CH2Ch with 2% NH3 MeOH = 0-40%) to afford (R)-N-(2-(4-((6-((5-fluoro-4-(4-fluoro-1-isopropyl-2-methyl-1H-benzo[d]imidazol-6-yl)pyrimidin-2-yl)amino)pyridin-3-yl)methyl)-2-methylmorpholin-2-yl)ethyl)-N-methylacetamide as a light orange oil. The oil was dissolved in water containing methanesulfonic acid (1.05 equiv.) and the solution was lyophilized to yield 17.2 mg of the mesylate salt as a tan solid.

Mesylate salt spectra

Reported as a mixture of amide rotamers 1H NMR (400 MHz, DMSO-d6) 88.70 (d, J = 3.8 Hz, 1H), 8.30 (s, 1H), 8.28 - 8.20 (m, 2H), 7.73 - 7.65 (m, 2H), 4.85 (pd, J = 6.8, 2.2 Hz, 1H), 3.74 -3.75 (m, 2H), 3.39 -3.12 (m, 4H), 2.91 (s, 1.5H), 2.76 (s, 1.5H), 2.65 (s, 3H), 2.44 -2.19 (m, 7H), 2.16 -2.04 (m, 1H), 1.97 (s, 1.5H), 1.94 (s, 1.5H), 1.63 (d, J = 6.9 Hz, 6H), 1.55 - 1.43 (m, 1H), 1.15 (s, 1.5H), 1.12 (s, 1.5H). MS: [M+H]+m/z 593.3.

Compound A54

To a cooled (0 °C) solution of (R)-2-(4-((6-((5-fluoro-4-(4-fluoro-1-isopropyl-2-methyl-1H-benzo[d]imidazol-6-yl)pyrimidin-2-yl)amino)pyridin-3-yl)methyl)-2-methylmorpholin-2-yl)ethan-1-ol (30 mg, 0.056 mmol) in DMF (0.6 mL) was added sodium hydride (5 mg, 0.11 mmol). After stirring for 30 min, the mixture was cooled to 0 °C and iodoethane (7 pL, 0.084 mmol) was added. The mixture was warmed to room temperature and stirred for 15 h. Upon completion, water (3 mL) was added and the mixture was extracted with CHCl3 (3 x 2 mL). The organic layers were combined and dried over Na2SO4, and concentrated in vacuo. The crude compound was purified using flash chromatography on SO2 (gradient elution of CH2Ch with 2% NH3-MeOH = 0-40%) to afford (R)-N-(5-((2-(2-ethoxyethyl)-2-methylmorpholino)methyl)pyridin-2-yl)-5-fluoro-4-(4-fluoro-1-isopropyl-2-methyl-1H-benzo[d]imidazol-6-yl)pyrimidin-2-amine as a light orange oil. The oil was dissolved in water containing methanesulfonic acid (1.05 equiv.) and the solution was lyophilized to yield 15.0 mg of the mesylate salt as a tan solid.

Mesylate salt spectra

1H NMR (400 MHz, DMSO-da) 88.65 (s, 1H), 8.41 - 8.29 (m, 1H), 8.15 (s, 1H), 7.96 - 7.52 (m, 3H), 4.81 (p, J = 7.0 Hz, 1H), 4.48 -4.17 (m, 3H), 3.71 -3.51 (m, 2H), 2.63 (s, 3H), 2.44 -2.12 (m, 8H), 2.03 - 1.87 (m, 1H), 1.76 - 1.43 (m, 8H), 1.42 - 1.06 (m, 7H). MS: [M+H]+ m/z 566.3.

Compound A12

To a cooled (0 °C) solution of (R)-2-(4-((6-((5-fluoro-4-(4-fluoro-1-isopropyl-2-methyl-1H-benzo[d]imidazol-6-yl)pyrimidin-2-yl)amino)pyridin-3-yl)methyl)-2-methylmorpholin-2-yl)acetaldehyde (30 mg, 0.056 mmol) in CHCh/MeOH (2:1, 1 mL) was added 3,3-difluoroazetidine^HCl (37 mg, 0.28 mmol). After 15 min of stirring, sodium triacetoxyborohydride (24 mg, 0.11 mmol) was added and the reaction was warmed to room temperature and stirred for 4 h. After completion, sodium hydroxide solution (0.5 M aq., 3 mL) was added and the mixture was extracted with CHCl (3 x 2 mL). The organic layers were combined and dried over NaSO, and concentrated in vacuo. The crude compound was purified using flash chromatography on SO (gradient elution of CHCl with 2% NHMeOH = 0-40%) to afford (R)-N-(5-((2-(2-(3,3-difluoroazetidin-1-yl)ethyl)-2-methylmorpholino)methyl)pyridin-2-yl)-5-fluoro-4-(4-fluoro-1-isopropyl-2-methyl-1H-benzo[d]imidazol-6-yl)pyrimidin-2-amine as a light orange oil. The oil was dissolved in water containing methanesulfonic acid (1.05 equiv.) and the solution was lyophilized to yield 21.8 mg of the mesylate salt as a white solid.

Mesylate salt spectra

1H NMR (400 MHz, DMSO-d6) 88.70 (d, J = 3.8 Hz, 1H), 8.31 (d, J = 1.4 Hz, 1H), 8.25 (d, J = 8.5 Hz, 1H), 8.22 (d, J = 2.2 Hz, 1H), 7.70 (m, 2H), 4.86 (p, J = 6.9 Hz, 1H), 3.59 (t, J = 5.0 Hz, 2H), 3.54 - 3.45 (m, 7H), 2.65 (s, 3H), 2.40 -2.25 (m, 6H), 2.21 (d, J = 11.2 Hz, 1H), 2.10 (d, J = 11.2 Hz, 1H), 1.85 - 1.69 (m, 1H), 1.64 (d, J = 6.8 Hz, 6H), 1.45 - 1.35 (m, 1H), 1.12 (s, 3H). MS: [M+H]+ m/z 613.3.

Compound A9

To a cooled (0 °C) solution of (R)-2-(4-((6-((5-fluoro-4-(4-fluoro-1-isopropyl-2-methyl-1H-benzo[d]imidazol-6-yl)pyrimidin-2-yl)amino)pyridin-3-yl)methyl)-2-methylmorpholin-2-yl)acetaldehyde (30 mg, 0.056 mmol) in CHCl3/MeOH (2:1, 1 mL) was added MgSO4 (40 mg) followed by 2,2,2-trifluoroethylamine (28 mg, 0.28 mmol). After warming to room temperature and stirring for 15 h, sodium borohydride (5 mg, 0.11 mmol) was added and the reaction was stirred for 1 h. After completion, water (3 mL) and brine (1 mL) were added and the mixture was extracted with CHCl (3 x 2 mL). The organic layers were combined and dried over NaSO, and concentrated in vacuo. The crude compound was purified using flash chromatography on SIO (gradient elution of CHCl with 2% NHMeOH = 0-40%) to afford (R)-5-fluoro-4-(4-fluoro-1-isopropyl-2-methyl-1H-benzo[d]imidazol-6-yl)-N-(5-((2-methyl-2-(2-((2,2,2-trifluoroethyl)amino)ethyl)morpholino)methyl)pyridin-2-yl)pyrimidin-2-amine as a light orange oil. The oil was dissolved in water containing methanesulfonic acid (1.05 equiv.) and the solution was lyophilized to yield 15.0 mg of the mesylate salt as a tan solid.

Mesylate salt spectra

1H NMR (400 MHz, DMSO-da) 88.70 (d, J = 3.8 Hz, 1H), 8.30 (d, J = 1.3 Hz, 1H), 8.28 - 8.19 (m, 2H), 7.73 -7.66 (m, 2H), 4.85 (p, J = 6.8 Hz, 1H), 3.60 (t, J = 4.8 Hz, 2H), 3.16 (q, J = 10.3 Hz, 2H), 2.66 -2.54 (m, 6 H), 2.37 -2.29 (m, 6H), 2.19 (d, J = 11.1 Hz, 1H), 2.11 (d, J = 11.1 Hz, 1H), 1.95 - 1.78 (m, 1H), 1.63 (d, J = 6.8 Hz, 6H), 1.48 (ddd, J = 13.5, 9.7, 6.1 Hz, 1H), 1.12 (s, 3H). MS: [M+H]+ m/z 619.3.

Compound A10

To a cooled (0 °C) solution of (R)-2-(4-((6-((5-fluoro-4-(4-fluoro-1-isopropyl-2-methyl-1H-benzo[d]imidazol-6-yl)pyrimidin-2-yl)amino)pyridin-3-yl)methyl)-2-methylmorpholin-2-yl)acetaldehyde (30 mg, 0.056 mmol) in CHCl3/MeOH (2:1, 1 mL) was added 3-hydroxy-3-methylazetidine^HCl (35 mg, 0.28 mmol). After 15 min of stirring, sodium triacetoxyborohydride (24 mg, 0.11 mmol) was added and the reaction was warmed to room temperature and stirred for 4 h. Upon completion, sodium hydroxide solution (0.5 M aq., 3 mL) was added and the mixture was extracted with CHCl (3 x 2 mL). The organic layers were combined and dried over NaSO, and concentrated in vacuo. The crude compound was purified using flash chromatography on SO (gradient elution of CHCl with 2% NHMeOH = 0-40%) to afford (R)-1-(2-(4-((6-((5-fluoro-4-(4-fluoro-1-isopropyl-2-methyl-1H-benzo[d]imidazol-6-yl)pyrimidin-2-yl)amino)pyridin-3-yl)methyl)-2-methylmorpholin-2-yl)ethyl)-3-methylazetidin-3-ol as a light orange oil. The oil was dissolved in water containing methanesulfonic acid (1.05 equiv.) and the solution was lyophilized to yield 15.9 mg of the mesylate salt as a tan solid.

Mesylate salt spectra

1H NMR (400 MHz, DMSO-d6) 88.70 (d, J = 3.8 Hz, 1H), 8.29 (d, J = 1.3 Hz, 1H), 8.27 - 8.19 (m, 2H), 7.74 -7.65 (m, 2H), 4.85 (hept, J = 6.8 Hz, 1H), 1.94 -1.78 (m, 1H), 1.63 (dd, J = 6.8 , 6H), 1.46 - 1.30 (m, 4H), 1.12 (s, 3H). MS: [M+H]+m/z 607.3.

Compound A21

To a cooled (0 °C) solution of (R)-2-(4-((6-((5-fluoro-4-(4-fluoro-1-isopropyl-2-methyl-1H-benzo[d]imidazol-6-yl)pyrimidin-2-yl)amino)pyridin-3-yl)methyl)-2-methylmorpholin-2-yl)acetaldehyde (30 mg, 0.056 mmol) in CHCl3 (0.7 mL) was added MgSO4 (20 mg) followed by ammonia (7 M in MeOH, 0.16 mL, 1.12 mmol). After warming to room temperature and stirring for 15 h, sodium borohydride (5 mg, 0.11 mmol) was added and the reaction was stirred for 4 h. Upon completion, water (3 mL) and brine (1 mL) were added and the mixture was extracted with CHCl (3 x 2 mL). The organic layers were combined and dried over NaSO, and concentrated in vacuo. The crude compound was purified using flash chromatography on SO (gradient elution of CHCl with 2% NHMeOH = 0-40%) to afford (R)-N-(5-((2-(2-aminoethyl)-2-methylmorpholino)methyl)pyridin-2-yl)-5-fluoro-4-(4-fluoro-1-isopropyl-2-methyl-1H-benzo[d]imidazol-6-yl)pyrimidin-2-amine as a light orange oil. The oil was dissolved in water containing methanesulfonic acid (1.05 equiv.) and the solution was lyophilized to yield 3.9 mg of the mesylate salt as a tan solid.

Mesylate salt spectra

1H NMR (400 MHz, DMSO-da) 88.70 (d, J = 3.8 Hz, 1H), 8.29 (d, J = 1.4 Hz, 1H), 8.26 - 8.19 (m, 2H), 7.74 -7.65 (m, 2H), 4.85 (p, J = 6.9 Hz, 1H), 3.61 (t, J = 5.0 Hz, 2H), 2.66 - 2.57 (m, 5H), 2.39 - 2.26 (m, 7H), 2.18 (d, J= 11.2 Hz, 1H), 2.10 (d, J = 11.2 Hz, 1H), 2.03 - 1.87 (m, 1H), 1.63 (d, J = 6.9 Hz, 6H), 1.47 (dt, J = 13.5, 8.0 Hz, 1H), 1.12 (s, 3H). MS: [M+H]+ m/z 537.2.

Compound A13

To a cooled (0 °C) solution of (R)-2-(4-((6-((5-fluoro-4-(4-fluoro-1-isopropyl-2-methyl-1H-benzo[d]imidazol-6-yl)pyrimidin-2-yl)amino)pyridin-3-yl)methyl)-2-methylmorpholin-2-yl)acetaldehyde (60 mg, 0.11 mmol) in CHCh/MeOH (2:1, 2 mL) was added 3-fluoroazetidine^HCl (63 mg, 0.56 mmol). After 15 min of stirring, sodium triacetoxyborohydride (48 mg, 0.22 mmol) was added and the reaction was warmed to room temperature and stirred for 3 h. Upon completion, sodium hydroxide solution (0.5 M aq., 6 mL) was added and the mixture was extracted with CHCl (3 x 4 mL). The organic layers were combined and dried over NaSO, and concentrated in vacuo. The crude compound was purified using flash chromatography on SO (gradient elution of CHCl with 2% MeOH/NH = 0-40%) to afford (R)-1-(2-(4-((6-((5-fluoro-4-(4-fluoro-1-isopropyl-2-methyl-1H-benzo[d]imidazol-6-yl)pyrimidin-2-yl)amino)pyridin-3-yl)methyl)-2-methylmorpholin-2-yl)ethyl)-3-methylazetidin-3-ol as a light orange oil. The oil was dissolved in water containing methanesulfonic acid (1.05 equiv.) and the solution was lyophilized to yield 44.8 mg of the mesylate salt as a white solid.

Mesylate salt spectra

1H NMR (400 MHz, DMSO-d6) 88.69 (d, J = 3.9 Hz, 1H), 8.30 (d, J = 1.3 Hz, 1H), 8.24 (dd, J = 8.5, 0.8 Hz, 1H), 8.22 - 8.17 (m, 1H), 7.74 - 7.65 (m, 2H), 5.09 (dddd, J = 57.7, 10.3, 5.7, 4.7 Hz, 1H), 4.85 (p, J = 6.9 Hz, 1H), 3.58 (t, J = 5.1 Hz, 2H), 3.48 - 3.32 (m, 2H), 3.04 - 2.89 (m, 2H), 2.64 (s, 3H), 2.42 - 2.25 (s, 8H), 2.20 (d, J = 11.1 Hz, 1H), 2.08 (d, J = 11.1 Hz, 1H), 1.77 - 1.60 (m, 8H), 1.44 - 1.31 (m, 1H), 1.10 (s, 3H). MS: [M+H]+ m/z 595.3.

Compound A15

To a cooled (0 °C) solution of (R)-2-(4-((6-((5-fluoro-4-(4-fluoro-1-isopropyl-2-methyl-1H-benzo[d]imidazol-6-yl)pyrimidin-2-yl)amino)pyridin-3-yl)methyl)-2-methylmorpholin-2-yl)acetaldehyde (45 mg, 0.084 mmol) in CHCl3/MeOH (2:1, 1.5 mL) was added (R)-(-)-3-fluoropyrrolidine • HCl (53 mg, 0.42 mmol). After 15 min of stirring, sodium triacetoxyborohydride (36 mg, 0.17 mmol) was added and the reaction was warmed to room temperature and stirred for 3 h. Upon completion, sodium hydroxide solution (0.5 M aq., 5 mL) was added and the mixture was extracted with CHCl (3 x 3 mL). The organic layers were combined and dried over NaSO, and concentrated in vacuo. The crude compound was purified using flash chromatography on SiO (gradient elution of CHCl with 2% NHMeOH = 0-40%) to afford 5-fluoro-4-(4-fluoro-1-isopropyl-2-methyl-1H-benzo[d]imidazol-6-yl)-N-(5-(((R)-2-(2-((R)-3-fluoropyrrolidin-1-yl)ethyl)-2-methylmorpholino)methyl)pyridin-2-yl)pyrimidin-2-amine as a light orange oil. The oil was dissolved in water containing methanesulfonic acid (1.05 equiv.) and the solution was lyophilized to yield 32.3 mg of the mesylate salt as a tan solid.

Mesylate salt spectra

1H NMR (400 MHz, DMSO-da) 88.73 - 8.67 (m, 1H), 8.30 (d, J = 1.3 Hz, 1H), 8.25 (d, J = 8.6 Hz, 1H), 8.22 (d, J = 2.1 Hz, 1H), 7.74 - 7.66 (m, 2H), 5.30 - 5.03 (m, 1H), 4.85 (hept, J = 6.9 Hz, 1H), 3.59 (d, J = 5.1 Hz, 2H), 2.98 -2.59 (m, 6H), 2.55 -2.00 (m, 13H), 1.99 - 1.72 (n, 2H), 1.69 - 1.49 (m, 7H), 1.12 (s, 3H). MS: [M+H]+ m/z 609.3.

Compound A14

To a cooled (0 °C) solution of (R)-2-(4-((6-((5-fluoro-4-(4-fluoro-1-isopropyl-2-methyl-1H-benzo[d]imidazol-6-yl)pyrimidin-2-yl)amino)pyridin-3-yl)methyl)-2-methylmorpholin-2-yl)acetaldehyde (45 mg, 0.084 mmol) in CHCh/MeOH (2:1, 1.5 mL) was added (S)-(+)-3-fluoropyrrolidine • HCl (53 mg, 0.42 mmol). After 15 min of stirring, sodium triacetoxyborohydride (36 mg, 0.17 mmol) was added and the reaction was warmed to room temperature and stirred for 3 h. Upon completion, sodium hydroxide solution (0.5 M aq., 5 mL) was added and the mixture was extracted with CHCl (3 x 3 mL). The organic layers were combined and dried over NaSO, and concentrated in vacuo. The crude compound was purified using flash chromatography on SO (gradient elution of CHCl with 2% NHMeOH = 0-40%) to afford 5-fluoro-4-(4-fluoro-1-isopropyl-2-methyl-1H-benzo[d]imidazol-6-yl)-N-(5-(((R)-2-(2-((S)-3-fluoropyrrolidin-1-yl)ethyl)-2-methylmorpholino)methyl)pyridin-2-yl)pyrimidin-2-amine as a light orange oil. The oil was dissolved in water containing methanesulfonic acid (1.05 equiv.) and the solution was lyophilized to yield 33.7 mg of the mesylate salt as a tan solid.

Mesylate salt spectra

1H NMR (400 MHz, DMSO-d6) 88.70 (d, J = 3.8 Hz, 1H), 8.31 (d, J = 1.3 Hz, 1H), 8.25 (dt, J = 8.5, 0.8 Hz, 1H), 8.22 (d, J = 2.2 Hz, 1H), 7.74 - 7.66 (m, 2H), 5.15 (dt, J = 55.9, 5.9 Hz, 1H), 4.85 (hept, J = 6.9 Hz, 1H), 3.60 (t, J = 4.8 Hz, 2H), 2.86 - 2.71 (m, 2H), 2.65 (s, 3H), 2.53 - 2.17 (m, 11H), 2.17 -1.98 (m, 2H), 1.97 -1.71 (m, 2H), 1.71 -1.51 (m, 8H), 1.12 (s, 3H). MS: [M+H]+ m/z 609.3.

Compound A6

To a solution of (R)-2-(4-((6-((5-fluoro-4-(4-fluoro-1-isopropyl-2-methyl-14-benzo[d]imidazol-6-yl)pyrimidin-2-yl)amino)pyridin-3-yl)methyl)-2-methylmorpholin-2-yl)acetaldehyde (45 mg, 0.084 mmol) in CHCl (1 mL) was added 2,2-difluoroethylamine (30 j L, 0.42 mmol) followed by acetic acid (24 j L, 0.84 mmol). After 10 min of stirring, sodium triacetoxyborohydride (36 mg, 0.17 mmol) was added and the reaction was stirred for 18 h. Upon completion, sodium bicarbonate solution (sat. aq., 5 mL) was added and the mixture was extracted with CHCl (3 x 3 mL). The organic layers were combined and dried over NaSO, and concentrated in vacuo. The crude compound was purified using flash chromatography on SiO (gradient elution of CHCh with 2% NHMeOH = 0-25%) to afford (R)-N-(5-((2-(2-((2,2-difluoroethyl)amino)ethyl)-2-methylmorpholino)methyl)pyridin-2-yl)-5-fluoro-4-(4-fluoro-1-isopropyl-2-methyl-1H-benzo[d]imidazol-6-yl)pyrimidin-2-amine as a light orange oil. The oil was dissolved in water containing methanesulfonic acid (1.05 equiv.) and the solution was lyophilized to yield 35.5 mg of the mesylate salt as a tan solid.

Mesylate salt spectra

1H NMR (400 MHz, DMSO-da) 88.70 (d, J = 3.8 Hz, 1H), 8.30 (d, J = 1.3 Hz, 1H), 8.27 - 8.19 (m, 2H), 7.73 -7.66 (m, 2H), 6.01 (tt, J = 56.0, 4.1 Hz, 1H), 4.86 (p, J = 6.9 Hz, 1H), 3.60 (t, J = 4.8 Hz, 2H), 2.93 (t, J = 16.2 Hz, 2H), 2.67 -2.57 (m, 5H), 2.32 (s, 6H), 2.20 (d, J = 11.2 Hz, 1H), 2.11 (d, J = 11.2 Hz, 1H), 2.01 - 1.86 (m, 1H), 1.63 (d, J = 6.9 Hz, 6H), 1.56 - 1.44 (m, 1H), 1.12 (s, 3H). MS: [M+H]+ m/z 601.3.

Compound A29

To a solution of 5-fluoro-4-(4-fluoro-1-isopropyl-2-methyl-1H-benzo[d]imidazol-6-yl)-N-(5-(((R)-2-(2-((R)-3-fluoropyrrolidin-1-yl)ethyl)-2-methylmorpholino)methyl)pyridin-2-yl)pyrimidin-2- (30 mg, 0.05 mmol) in CHCl/MeOH (2:1, 1 mL) was added formaldehyde (37% in H2O, 5<j>L, 0.06 mmol). After 5 min of stirring, sodium triacetoxyborohydride (22 mg, 0.10 mmol) was added and the reaction was stirred for 2 h. Upon completion, sodium bicarbonate solution (sat. aq., 5 mL) was added and the mixture was extracted with CHCl (3 x 3 mL). The organic layers were combined and dried over NaSO, and concentrated in vacuo. The crude compound was purified using flash chromatography on SO (gradient elution of CHCl with 2% NHMeOH = 0.10%) to afford (R)-N-(5-((2-(2-((2,2-difluoroethyl)(methyl)amino)ethyl)-2-methylmorpholino)methyl)pyridin-2-yl)-5-fluoro-4-(4-fluoro-1-isopropyl-2-methyl-1H-benzo[d]imidazol-6-yl)pyrimidin-2-amine as a light orange oil. The oil was dissolved in water containing methanesulfonic acid (1.05 equiv.) and the solution was lyophilized to yield 25.2 mg of the mesylate salt as a tan solid.

Mesylate salt spectra

1H NMR (400 MHz, DMSO-d6) 88.70 (d, J = 3.8 Hz, 1H), 8.30 (d, J = 1.4 Hz, 1H), 8.27 - 8.20 (m, 2H), 7.74 -7.65 (m, 2H), 6.06 (t, J = 55.8 Hz, 1H), 4.85 (hept, J = 6.8 Hz, 1H), 3.67 - 3.54 (m, 2H), 2.83 - 2.60 (s, 6H), 2.49 -2.17 (m, 12H), 2.17 -2.03 (m, 1H), 1.95 - 1.77 (m, 1H), 1.63 (d, J = 6.9Hz, 6H), 1.61 -1.50 (m, 1H), 1.12 (s, 3H). MS: [M+H]+ m/z 615.3.

Compound A20

To a cooled (0 °C) solution of allyl 4-benzoyl-3-oxomorpholine-2-carboxylate (2.0 g, 6.9 mmol) in DMF (7 mL) was added iodoethane (1.45 mL, 13.8 mmol), followed by cesium carbonate (4.5 g, 13.8 mmol). The reaction was stirred for 2 hours, at which time saturated ammonium chloride (aq., 20 mL) and water (20 mL) were added. The mixture was extracted with Et2O (3 x 20 mL), and the combined organic layers were dried over Na2SO4 and concentrated in vacuo. The crude compound was purified using flash chromatography on SO 2 (gradient elution of hexanes with EtOAc = 0-50%) to provide allyl 4-benzoyl-2-ethyl-3-oxomorpholine-2-carboxylate (1.7 g, 78% yield) as a white solid.

MS: [M+H]+ m/z 317.1.

Pd(OAc) (6 mg, 0.027 mmol), (R)-(p-CF)-t-BuPHOX (80 mg, 0.134 mmol), and MTBE (40 mL) were added to a Schlenk flask equipped with a stir bar. The mixture was stirred for 30 min, after which allyl 4-benzoyl-2-ethyl-3-oxomorpholine-2-carboxylate (1.7 g, 5.4 mmol) was added as a solution in MTBE (15 mL). The flask was sealed and heated at 55 °C for 4 days. Once complete, the reaction was cooled to room temperature and vented to release the evolved CO. The mixture was filtered through Celite, washed with EtOAc, and concentrated in vacuo. The crude compound was purified using flash chromatography on SiO2 (gradient elution of hexanes with EtOAc = 0-20%) to provide (R)-2-allyl-4-benzoyl-2-ethylmorpholin-3-one (1.3 g, 90% yield, 98% ee) as a white solid.

MS: [M+H]+ m/z 274.1.

To a cooled (0 °C) solution of (R)-2-allyl-4-benzoyl-2-ethylmorpholin-3-one (1.3 g, 4.8 mmol) in iPrOH (100 mL) was added lithium hydroxide (4 M in H2O, 1.8 mL, 7.1 mmol). The reaction was warmed to room temperature and stirred for 8 h. Upon completion, MgSO4 was added, and the mixture was filtered through Celite, washed with CH2Cl2, and concentrated in vacuo. The crude compound was purified using flash chromatography on SiO2 (gradient elution of CH2Ch with 2% NH3-MeOH = 0-30%) to afford (R)-2-allyl-2-ethylmorpholin-3-one (367 mg, 45% yield) as a colorless oil.

MS: [M+H]+ m/z 170.1.

To a cooled (0 °C) solution of (R)-2-allyl-2-ethylmorpholin-3-one (367 mg, 2.2 mmol) in THF (11 mL) was added lithium aluminum hydride (250 mg, 6.5 mmol). The mixture was warmed to room temperature and then to 60 °C for 2 h. Upon completion, the mixture was cooled to 0 °C, and Et2O (11 mL) was added. Water (0.3 mL) was slowly added, followed by sodium hydroxide solution (1 M aq., 0.3 mL), and the mixture was warmed to room temperature and stirred for 15 min. The mixture was dried with MgSO4, filtered through Celite, and concentrated in vacuo to afford (R)-2-allyl-2-ethylmorpholine (336 mg) as a colorless oil. The compound was used immediately without further purification.

MS: [M+H]+ m/z 156.2.

To a solution of (R)-2-allyl-2-ethylmorpholine (336 mg, 2.2 mmol) and 6-((5-fluoro-4-(4-fluoro-1-isopropyl-2-methyl-1H-benzo[d]imidazol-6-yl)pyrimidin-2-yl)amino)nicotinaldehyde (739 mg, 1.8 mmol) in DMSO (18 mL) was added sodium triacetoxyborohydride (770 mg, 3.6 mmol), followed by acetic acid (1.1 mL, 18 mmol). The reaction was heated to 60 °C and stirred for 24 h. Upon completion, the mixture was cooled to 0 °C, and aqueous sodium hydroxide solution (1 M aq., 40 mL) was added, followed by water (15 mL). The resulting heterogeneous mixture was stirred for 1 h, then filtered to collect the solid, rinsed with cold water and finally lyophilized to yield (R)-N-(5-((2-allyl-2-ethylmorpholino)methyl)pyridin-2-yl)-5-fluoro-4-(4-fluoro-1-isopropyl-2-methyl-1H-benzo[d]imidazol-6-yl)pyrimidin-2-amine (862 mg) as a grey solid. The compound was used without further purification.

MS: [M+H]+ m/z 548.3.

To a solution of (R)-N-(5-((2-allyl-2-ethylmorpholino)methyl)pyridin-2-yl)-5-fluoro-4-(4-fluoro-1-isopropyl-2-methylMH-benzo[d]imidazol-6-yl)pyrimidin-2-amine (862 mg, 1.6 mmol) in THF/H2O (2:1, 39 mL) was added osmium tetroxide (4% in H2O, 0.5 mL, 0.08 mmol), followed by sodium periodate (1.00 g, 4.7 mmol). The reaction was sonicated for 2.5 h and then stirred for 4.5 h. Upon completion, saturated sodium thiosulfate (aq, 80 mL) was added, followed by sodium hydroxide solution (1 M aq, 50 mL), and the mixture was extracted with CHCl (3 x 50 mL). The organic layers were combined and dried over Na2SO4, and concentrated in vacuo to afford (R)-2-(2-ethyl-4-((6-((5-fluoro-4-(4-fluoro-1-isopropyl-2-methyl-1H-benzo[d]imidazol-6-yl)pyrimidin-2-yl)amino)pyridin-3-yl)methyl)morpholin-2-yl)acetaldehyde (815 mg) as a black solid. The compound was used without further purification.

MS: [M+H]+ m/z 550.3.

To a cooled (0 °C) solution of (R)-2-(2-ethyl-4-((6-((5-fluoro-4-(4-fluoro-1-isopropyl-2-methyl-1H-benzo[d]imidazol-6-yl)pyrimidin-2-yl)amino)pyridin-3-yl)methyl)morpholin-2-yl)acetaldehyde (70 mg, 0.13 mmol) in CHCh/MeOH (2:1, 2 mL) was added dimethylamine (2 M in THF, 0.64 mL, 1.3 mmol). After 15 min of stirring, sodium triacetoxyborohydride (55 mg, 0.25 mmol) was added and the reaction was warmed to room temperature and stirred for 2 h. Upon completion, sodium hydroxide solution (0.5 M aq., 6 mL) was added and the mixture was extracted with CHCl (3 x 3 mL). The organic layers were combined and dried over Na2SO4, and concentrated in vacuo. The crude compound was purified using flash chromatography on SO2 (gradient elution of CH2Cl2 with 2% NH3 MeOH = 0-40%) to afford (R)-N-(5-((2-(2-(dimethylamino)ethyl)-2-ethylmorpholino)methyl)pyridin-2-yl)-5-fluoro-4-(4-fluoro-1-isopropyl-2-methyl-1H-benzo[d]imidazol-6-yl)pyrimidin-2-amine as a light orange oil. The oil was dissolved in water containing methanesulfonic acid (1.05 equiv.) and the solution was lyophilized to yield 25.3 mg of the mesylate salt as a tan solid.

Mesylate salt spectra

1H NMR (400 MHz, DMSO-da) 88.70 (d, J = 3.8 Hz, 1H), 8.30 (d, J = 1.3 Hz, 1H), 8.27 - 8.19 (m, 2H), 7.73 -7.65 (m, 2H), 4.85 (hept, J = 6.9 Hz, 1H), 3.59 (m, 2H), 2.65 (s, 3H), 2.42 - 2.03 (m, 17H), 1.85 - 1.71 (m, 1H), 1.70 -1.51 (m, 8H), 1.49 - 1.36 (m, 1H), 0.74 (t, J = 7.5 Hz, 3H). MS: [M+H]+ m/z 579.3.

Compound A3

To a cooled (0 °C) solution of (R)-2-(4-((6-((5-fluoro-4-(4-fluoro-1-isopropyl-2-methyl-1H-benzo[d]imidazol-6-yl)pyrimidin-2-yl)amino)pyridin-3-yl)methyl)-2-methylmorpholin-2-yl)acetaldehyde (303 mg, 0.57 mmol) in CHCh/MeOH (2:1, 7 mL) was added diethylamine (0.3 mL, 2.83 mmol). After 15 min of stirring, sodium triacetoxyborohydride (242 mg, 1.14 mmol) was added and the reaction was warmed to room temperature and stirred for 2 h. Upon completion, sodium hydroxide solution (0.5 M aq., 14 mL) was added and the mixture was extracted with CHCl (3 x 10 mL). The organic layers were combined and dried over NaSO, and concentrated in vacuo. The crude compound was purified using flash chromatography on SO (gradient elution of CHCl with 2% NHMeOH = 0-40%) to afford (S)-N-(5-((2-(2-(diethylamino)ethyl)-2-methylmorpholino)methyl)pyridin-2-yl)-5-fluoro-4-(4-fluoro-1-isopropyl-2-methyl-1H-benzo[d]imidazol-6-yl)pyrimidin-2-amine as a light orange oil. The oil was dissolved in water containing methanesulfonic acid (1.05 equiv.) and the solution was lyophilized to yield 230 mg of the mesylate salt as a white solid.

Mesylate salt spectra

1H NMR (400 MHz, DMSO-d6) 88.70 (d, J = 3.9 Hz, 1H), 8.30 (d, J = 1.3 Hz, 1H), 8.24 (dd, J = 8.5, 0.8 Hz, 1H), 8.21 (dd, J = 2.4, 0.8 Hz, 1H), 7.74 - 7.65 (m, 2H), 4.86 (p, J = 6.8 Hz, 1H), 3.60 (t, J = 4.8 Hz, 2H), 2.71 - 2.51 (m, 4H), 2.47 - 2.26 (m, 12H), 2.23 (d, J = 11.0 Hz, 1H), 2.08 (d, J = 11.2Hz, 1H), 1.87 - 1.72 (m, 1H), 1.63 (d, J = 6.9 Hz, 6H), 1.53 (ddd, J= 13.3, 10.3, 5.9 Hz, 1H), 1.11 (s, 3H), 0.90 (t, J = 7.1 Hz, 6H). MS: [M+H]+ m/z 593.3.

Compound A16

To a cooled (0 °C) solution of (R)-2-(4-((6-((5-fluoro-4-(4-fluoro-1-isopropyl-2-methyl-1H-benzo[d]imidazol-6-yl)pyrimidin-2-yl)amino)pyridin-3-yl)methyl)-2-methylmorpholin-2-yl)acetaldehyde (45 mg, 0.084 mmol) in CHCh/MeOH (2:1, 1.5 mL) was added pyrrolidine (35 pL, 0.42 mmol). After 15 min of stirring, sodium triacetoxyborohydride (36 mg, 0.17 mmol) was added and the reaction was warmed to room temperature and stirred for 2 h. Upon completion, sodium hydroxide solution (0.5 M aq., 5 mL) was added and the mixture was extracted with CHCl (3 x 3 mL). The organic layers were combined and dried over NaSO, and concentrated in vacuo. The crude compound was purified using flash chromatography on SO (gradient elution of CHCl with 2% NHMeOH = 0-40%) to afford (R)-5-fluoro-4-(4-fluoro-1-isopropyl-2-methyl-1H-benzo[d]imidazol-6-yl)-N-(5-((2-methyl-2-(2-(pyrrolidin-1-yl)ethyl)morpholino)methyl)pyridin-2-yl)pyrimidin-2-amine as a light orange oil. The oil was dissolved in water containing methanesulfonic acid (1.05 equiv.) and the solution was lyophilized to yield 29.7 mg of the mesylate salt as a white solid.

Mesylate salt spectra

1H NMR (400 MHz, DMSO-da) 88.70 (d, J = 3.8 Hz, 1H), 8.30 (d, J = 1.3 Hz, 1H), 8.24 (dd, J = 8.5, 0.8 Hz, 1H), 8.21 (d, J = 2.2 Hz, 1H), 7.74 - 7.65 (m, 2H), 4.85 (p, J = 6.9 Hz, 1H), 3.59 (t, J = 4.8 Hz, 2H), 2.64 (s, 4H), 2.47 - 2.26 (m, 12H), 2.23 (d, J = 11.1 Hz, 1H), 2.09 (d, J = 11.1 Hz, 1H), 1.99 - 1.78 (m, 1H), 1.69 - 1.53 (m, 11H), 1.11 (s, 3H). MS: [M+H]+ m/z 591.3.

Compound A11

To a cooled (0 °C) solution of (R)-2-(4-((6-((5-fluoro-4-(4-fluoro-1-isopropyl-2-methyl-1H-benzo[d]imidazol-6-yl)pyrimidin-2-yl)amino)pyridin-3-yl)methyl)-2-methylmorpholin-2-yl)acetaldehyde (45 mg, 0.084 mmol) in CHCh/MeOH (2:1, 1.5 mL) was added 2-methylaziridine (30 pL, 0.42 mmol). After 15 min of stirring, sodium triacetoxyborohydride (36 mg, 0.17 mmol) was added and the reaction was warmed to room temperature and stirred for 2 h. Upon completion, sodium hydroxide solution (0.5 M aq., 5 mL) was added and the mixture was extracted with CHCl (3 x 3 mL). The organic layers were combined and dried over NaSO, and concentrated in vacuo. The crude compound was purified using flash chromatography on SO (gradient elution of CHCl with 2% NHMeOH = 0-40%) to afford 5-fluoro-4-(4-fluoro-1-isopropyl-2-methyl-1H-benzo[d]imidazol-6-yl)-N-(5-(((2R)-2-methyl-2-(2-(2-methylaziridin-1-yl)ethyl)morpholino)methyl)pyridin-2-yl)pyrimidin-2-amine as a light orange oil. The oil was dissolved in water containing methanesulfonic acid (1.05 equiv.) and the solution was lyophilized to yield 15.1 mg of the mesylate salt as a tan solid.

Mesylate salt spectra

1H NMR (400 MHz, DMSO-d6) 88.70 (d, J = 3.8 Hz, 1H), 8.30 (d, J = 1.3 Hz, 1H), 8.28 - 8.14 (m, 2H), 7.81 -7.60 (m, 2H), 5.80 (ddt, J = 17.3, 10.3, 5.8 Hz, 1H), 5.13 (dq, J = 17.2, 1.7 Hz, 1H), 5.02 (ddt, J = 10.2, 2.4, 1.3 Hz, 1H), 4.85 (hept, J = 6.9 Hz, 1H), 3.60 (t, J = 5.0 Hz, 2H), 3.12 (dt, J = 5.9, 1.5 Hz, 2H), 2.70 - 2.60 (m, 4H), 2.47 (m, 2H), 2.38 -2.27 (m, 7H), 2.19 (d, J = 11.0 Hz, 1H), 2.10 (d, J = 11.0 Hz, 1H), 1.98 - 1.77 (m, 1H), 1.63 (d, J = 6.9 Hz, 6H), 1.50 (ddd, J = 13.6, 9.9, 6.1 Hz, 1H), 1.11 (s, 3H). MS: [M+H]+ m/z 577.3.

Compound A17

To a cooled (0 °C) solution of (R)-2-(4-((6-((5-fluoro-4-(4-fluoro-1-isopropyl-2-methyl-1H-benzo[d]imidazol-6-yl)pyrimidin-2-yl)amino)pyridin-3-yl)methyl)-2-methylmorpholin-2-yl)acetaldehyde (45 mg, 0.084 mmol) in CHCl3/MeOH (2:1, 1.5 mL) was added azetidine^HCl (40 mg, 0.42 mmol). After 15 min of stirring, sodium triacetoxyborohydride (36 mg, 0.17 mmol) was added and the reaction was warmed to room temperature and stirred for 2 h. Upon completion, sodium hydroxide solution (0.5 M aq., 5 mL) was added and the mixture was extracted with CHCl (3 x 3 mL). The organic layers were combined and dried over NaSO, and concentrated in vacuo. The crude compound was purified using flash chromatography on SiO (gradient elution of CHCl with 2% NHMeOH = 0-40%) to afford (R)-N-(5-((2-(2-(azetidin-1-yl)ethyl)-2-methylmorpholino)methyl)pyridin-2-yl)-5-fluoro-4-(4-fluoro-1-isopropyl-2-methyl-1H-benzo[d]imidazol-6-yl)pyrimidin-2-amine as a light orange oil. The oil was dissolved in water containing methanesulfonic acid (1.05 equiv.) and the solution was lyophilized to yield 29.7 mg of the mesylate salt as a white solid.

Mesylate salt spectra

1H NMR (400 MHz, DMSO-da) 88.70 (d, J = 3.8 Hz, 1H), 8.30 (d, J = 1.3 Hz, 1H), 8.24 (dd, J = 8.6, 0.9 Hz, 1H), 8.22 - 8.19 (m, 1H), 7.73 - 7.65 (m, 2H), 4.86 (p, J = 6.9 Hz, 1H), 3.58 (t, J = 4.9 Hz, 2H), 3.15 - 3.02 (m, 4H), 2.65 (s, 3H), 2.32 (s, 8H), 2.20 (d, J = 11.1 Hz, 1H), 2.08 (d, J = 11.3Hz, 1H), 1.92 (p, J = 6.9 Hz, 2H), 1.75 -1.57 (m, 8H), 1.40 - 1.29 (m, 1H), 1.09 (s, 3H). MS: [M+H]+ m/z 577.3.

Compound A18

To a cooled (0 °C) solution of (R)-2-(4-((6-((5-fluoro-4-(4-fluoro-1-isopropyl-2-methyl-1H-benzo[d]imidazol-6-yl)pyrimidin-2-yl)amino)pyridin-3-yl)methyl)-2-methylmorpholin-2-yl)acetaldehyde (45 mg, 0.084 mmol) in CHCh/MeOH (2:1, 1.5 mL) was added 3-methoxy-3-methylazetidine^HCl (58 mg, 0.42 mmol). After 15 min of stirring, sodium triacetoxyborohydride (36 mg, 0.17 mmol) was added and the reaction was warmed to room temperature and stirred for 2 h. Upon completion, sodium hydroxide solution (0.5 M aq., 5 mL) was added and the mixture was extracted with CHCl (3 x 3 mL). The organic layers were combined and dried over NaSO, and concentrated in vacuo. The crude compound was purified using flash chromatography on SO (gradient elution of CHCl with 2% NHMeOH = 0-40%) to afford (R)-5-fluoro-4-(4-fluoro-1-isopropyl-2-methyl-1H-benzo[d]imidazol-6-yl)-N-(5-((2-(2-(3-methoxy-3-methylazetidin-1-yl)ethyl)-2-methylmorpholino)methyl)pyridin-2-yl)pyrimidin-2-amine as a light orange oil. The oil was dissolved in water containing methanesulfonic acid (1.05 equiv.) and the solution was lyophilized to yield 32.8 mg of the mesylate salt as a tan solid.

Mesylate salt spectra

1H NMR (400 MHz, DMSO-d6) 88.70 (d, J = 3.8 Hz, 1H), 8.30 (d, J = 1.3 Hz, 1H), 8.25 (dd, J = 8.5, 0.8 Hz, 1H), 8.22 - 8.19 (m, 1H), 7.74 - 7.65 (m, 2H), 4.85 (p, J = 6.9 Hz, 1H), 3.58 (t, J = 4.9 Hz, 2H), 3.11 - 3.04 (m, 5H), 2.83 (d, J = 7.1 Hz, 2H), 2.64 (s, 3H), 2.43 - 2.25 (m, 8H), 2.21 (d, J = 11.1 Hz, 1H), 2.07 (d, J = 11.1 Hz, 1H), 1.77 - 1.58 (m, 7H), 1.44 - 1.26 (m, 5H), 1.10 (s, 3H). MS: [M+H]+ m/z 621.3.

Compounds A52 and A53

To a flask containing (S)-2-allyl-4-benzoyl-2-methylmorpholin-3-one (1.13 g, 4.4 mmol) was added PdCh(CHCN) (34 mg, 0.13 mmol) and p-benzoquinone (0.71 g, 6.5 mmol). The flask was evacuated and refilled with nitrogen gas three times before t-BuOH/MeOH (1:1, 22 mL) and HO (0.2 mL) were added. The reaction was heated to 50 °C and stirred for 4 h. Upon completion, the mixture was cooled to room temperature and the solvent was removed in vacuo. Sodium carbonate solution (1 M aq., 100 mL) was then added and the mixture was extracted using EtOAc (3 x 100 mL). The combined organic layers were dried over NaSO and concentrated in vacuo. The crude compound was purified using flash chromatography on SiO2 (gradient elution of hexanes with EtOAc = 0-30%) to provide (S)-4-benzoyl-2-methyl-2-(2-oxopropyl)morpholin-3-one (820 mg, 68% yield) as a colorless oil.

MS: [M+H]+ m/z 276.1.

To a cooled (0 °C) solution of (S)-4-benzoyl-2-methyl-2-(2-oxopropyl)morpholin-3-one (43 mg, 0.16 mmol) in THF (1.6 mL) was added lithium aluminum hydride (59 mg, 1.6 mmol). The mixture was warmed to room temperature and 50 °C for 22 h. Upon completion, the mixture was cooled to 0 °C and Et2O (2 mL) was added. Water (0.071 mL) was added slowly, followed by sodium hydroxide solution (1 M aq., 0.071 mL), and the mixture was warmed to room temperature and stirred for 15 min. The mixture was dried with MgSO4, filtered through Celite, and concentrated in vacuo to afford 1-((S)-2-methylmorpholin-2-yl)propan-2-ol (25 mg) as a colorless oil and a mixture of diastereoisomers in the alcohol. The compound was used immediately without further purification.

MS: [M+H]+ m/z 160.1.

To a solution of 1-((S)-2-methylmorpholin-2-yl)propan-2-ol (25 mg, 0.16 mmol) and 6-((5-fluoro-4-(4-fluoro-1-isopropyl-2-methyl-1H-benzo[d]imidazol-6-yl)pyrimidin-2-yl)amino)nicotinaldehyde (26 mg, 0.06 mmol) in DMSO (1.7 mL) was added sodium triacetoxyborohydride (41 mg, 0.19 mmol), followed by acetic acid (37 pL, 0.64 mmol). The reaction was heated to 60 °C and stirred for 16 h. Upon completion, the mixture was filtered and purified using preparative HPLC (gradient elution of H2O 0.25% TFA with MeCN = 10-24%) to afford 1-((S)-4-((6-((5-fluoro-4-(4-fluoro-1-isopropyl-2-methyl-1H-benzo[d]imidazol-6-yl)pyrimidin-2-yl)amino)pyridin-3-yl)methyl)-2-methylmorpholin-2-yl)propan-2-ol (8 mg major diastereomer, 6 mg minor diastereomer) as light brown solids.

Free base spectra

Compound A52: Peak 1, minor diastereomer

1H NMR (400 MHz, Chloroform-d) 88.47 (d, J = 3.7 Hz, 1H), 8.44 (dd, J = 8.6, 0.8 Hz, 1H), 8.36 (s, 1H), 8.31 -8.26 (m, 1H), 8.21 (d, J = 1.3 Hz, 1H), 7.82 (dd, J = 11.6, 1.4 Hz, 1H), 7.72 (dd, J = 8.6, 2.3 Hz, 1H), 4.76 (hept, J = 7.0 Hz, 1H), 4.25 (dtt, J = 12.8, 6.6, 3.2 Hz, 1H), 4.09 (s, 1H), 3.86 -3.77 (m, 2H), 3.45 (q, J = 12.2 Hz, 2H), 2.72 (s, 3H), 2.55 (dt, J = 10.6, 3.9 Hz, 1H), 2.41 - 2.29 (m, 2H), 2.15 (d, J = 11.2 Hz, 1H), 1.90 (dd, J = 14.7, 10.5 Hz, 1H), 1.74 (d, J = 7.0 Hz, 6H), 1.40 (s, 3H), 1.32 (dd, J = 14.5, 1.4 Hz, 1H), 1.17 (d, J = 6.1 Hz, 3H). MS: [M+H]+ m/z 552.3.

Compound A53: Peak 1, major diastereomer

1H NMR (400 MHz, Chloroform-d) 88.71 - 8.14 (m, 5H), 7.82 (dd, J = 11.5, 1.2 Hz, 1H), 7.73 (d, J = 8.6 Hz, 1H), 4.76 (hept, J = 6.9 Hz, 1H), 4.23 -4.10 (m, 1H), 3.88 (ddd, J = 12.2, 9.4, 2.8 Hz, 1H), 3.75 (dt, J = 11.9, 3.4 Hz, 1H), 3.56 (d, J = 13.1 Hz, 1H), 3.39 (d, J = 13.2 Hz, 1H), 2.72 (s, 3H), 2.59 (d, J = 11.3 Hz, 1H), 2.44 (d, J = 11.4 Hz, 1H), 2.38 -2.22 (m, 2H), 1.86 - 1.68 (m, 8H), 1.52 (dd, J = 14.9, 1.6 Hz, 1H), 1.35 (s, 3H), 1.16 (d, J = 6.2Hz, 3H). MS: [M+H]+ m/z 552.3.

C o m p u e s t A 47

To a cooled (0 °C) solution of allyl 4-benzoyl-1-(4-methoxybenzyl)-3-oxopiperazine-2-carboxylate (1.2 g, 3.0 mmol) in THF (30 mL) was added sodium hydride (60% dispersion in mineral oil, 0.14 g, 3.5 mmol). After 45 min of stirring, iodomethane (0.3 mL, 4.4 mmol) was added, and the reaction was warmed to room temperature, stirring for 20 h. Upon completion, saturated aqueous ammonium chloride (30 mL) and saturated aqueous sodium bicarbonate (30 mL) were added. The mixture was extracted with CH2Ch (2 x 50 mL), and the combined organic layers were dried over Na2SO4 and concentrated in vacuo. The crude compound was purified using flash chromatography on SiO2 (gradient elution of hexanes with EtOAc = 0-50%) to afford allyl 4-benzoyl-1-(4-methoxybenzyl)-2-methyl-3-oxopiperazine-2-carboxylate (141 mg, 11% yield) as a colorless oil.

MS: [M+H]+ m/z 423.2.

Pd(OAc) (3.7 mg, 0.017 mmol), (S)-(p-CF)-t-BuPHOX (25 mg, 0.041 mmol), and THF (7 mL) were added to a Schlenk flask equipped with a stir bar. The mixture was stirred for 30 min, after which allyl 4-benzoyl-1-(4-methoxybenzyl)-2-methyl-3-oxopiperazine-2-carboxylate (141 mg, 0.33 mmol) was added as a solution in THF (4 mL). The flask was sealed and heated at 50 °C for 3 days. Upon completion, the reaction was cooled to room temperature and vented to release the evolved CO. The mixture was filtered through Celite, washed with EtOAc, and concentrated in vacuo. The crude compound was purified using flash chromatography on SiO2 (gradient elution of hexanes with EtOAc = 0-30%) to afford (S)-3-allyl-1-benzoyl-4-(4-methoxybenzyl)-3-methylpiperazin-2-one (78 mg, 62% yield, 90% ee) as a colorless oil.

MS: [M+H]+ m/z 379.2.

To a cooled (0 °C) solution of (S)-3-allyl-1-benzoyl-4-(4-methoxybenzyl)-3-methylpiperazin-2-one (78 mg, 0.21 mmol) in THF (2.2 mL) was added lithium aluminum hydride (78 mg, 2.1 mmol). The mixture was warmed to room temperature and heated at 50 °C for 18 h. Upon completion, the mixture was cooled to 0 °C and Et2O (3 mL) was added. Water (0.1 mL) was slowly added, followed by sodium hydroxide solution (1 M aq., 0.1 mL), and the mixture was warmed to room temperature and stirred for 15 min. The mixture was dried with MgSO4, filtered through Celite, and concentrated in vacuo to afford (S)-2-allyl-4-benzyl-1-(4-methoxybenzyl)-2-methylpiperazine (18 mg) as a colorless oil. The compound was used immediately without further purification.

MS: [M+H]+ m/z 351.2.

To a solution of (S)-2-allyl-4-benzyl-1-(4-methoxybenzyl)-2-methylpiperazine (18 mg, 0.051 mmol) in EtOH/CH2Cl2 (4:1, 1.25 mL) was added 10% palladium on carbon (10 mg). The reaction vessel was placed on a pressure pump and charged with 1000 psi of hydrogen gas. The system was heated at 60 °C for 2 days. After completion, the mixture was filtered through Celite, washed with CH2Cl/MeOH (1:1), and concentrated in vacuo to provide (S)-2-methyl-2-propylpiperazine (7 mg) as a colorless oil. The compound was used immediately without further purification.

MS: [M+H]+ m/z 143.2.

To a solution of (S)-2-methyl-2-propylpiperazine (7 mg, 0.05 mmol) and 6-((5-fluoro-4-(4-fluoro-1-isopropyl-2-methyl-1H-benzo[d]imidazol-6-yl)pyrimidin-2-yl)amino)nicotinaldehyde (10 mg, 0.025 mmol) in DMSO (0.5 mL) was added sodium triacetoxyborohydride (16 mg, 0.075 mmol), followed by acetic acid (14 L, 0.25 mmol). The reaction was heated to 60 °C and stirred for 24 h. Upon completion, the mixture was filtered and purified using preparative HPLC (gradient elution of 0.1% H2O AcOH with MeCN = 10-50%) to afford (S)-5-fluoro-4-(4-fluoro-1-isopropyl-2-methyl-1H-benzo[d]imidazol-6-yl)-N-(5-((3-methyl-3-propylpiperazin-1-yl)methyl)pyridin-2-yl)pyrimidin-2-amine (10 mg) as a white solid.

Free base spectra

1H NMR (400 MHz, Chloroform-d) 88.46 (d, J = 3.7 Hz, 1H), 8.42 (dd, J = 8.6, 0.8 Hz, 1H), 8.31 - 8.24 (m, 2H), 8.23 (d, J = 1.3 Hz, 1H), 7.85 - 7.78 (m, 1H), 7.72 (dd, J = 8.6, 2.3 Hz, 1H), 4.76 (hept, J = 7.0 Hz, 1H), 3.45 (s, 2H), 2.95 (d, J = 5.4 Hz, 2H), 2.72 (s, 3H), 2.52 - 2.33 (m, 2H), 2.28 - 2.10 (m, 2H), 1.74 (d, J = 6.9 Hz, 6H), 1.68 - 1.49 (m, 1H), 1.47 -1.19 (m, 4H), 1.13 (s, 3H), 0.93 (t, J = 6.9 Hz, 3H). MS: [M+H]+ m/z 535.3.

Compound B3

To a cooled (0 °C) solution of (R)-2-(4-((6-((5-fluoro-4-(4-fluoro-1-isopropyl-2-methyl-1H-benzo[d]imidazol-6-yl)pyrimidin-2-yl)amino)pyridin-3-yl)methyl)-2-methylmorpholin-2-yl)acetaldehyde (B2) (30 mg, 0.056 mmol) in tBuOH/MeCN (1:1, 1 mL) was added 2-methyl-2-butene (0.12 mL, 1.12 mmol), followed by the dropwise addition of a solution of sodium chlorite (50 mg, 0.56 mmol) and sodium monophosphate (87 mg, 0.56 mmol) in water (1 mL). The reaction was warmed to room temperature and stirred for 5 h. Upon completion, water (1 mL) was added and the mixture was extracted with CHCl (3 x 2 mL). The organic layers were combined and dried over NaSO, and concentrated in vacuo to afford (R)-2-(4-((6-((5-fluoro-4-(4-fluoro-1-isopropyl-2-methyl-1H-benzo[d]imidazol-6-yl)pyrimidin-2-yl)amino)pyridin-3-yl)methyl)-2-methylmorpholin-2-yl)acetic acid

(31 mg) as a brown solid. The compound was used without further purification.

MS: [M+H]+ m/z 552.3.

C o m p u e s t A 55

To a cooled (0 °C) solution of (R)-2-(4-((6-((5-fluoro-4-(4-fluoro-1-isopropyl-2-methyl-1H-benzo[d]imidazol-6-yl)pyrimidin-2-yl)amino)pyridin-3-yl)methyl)-2-methylmorpholin-2-yl)acetic acid (31 mg, 0.056 mmol) in DMF (2.5 mL) was added N,N-diisopropylethylamine (50 L, 0.28 mmol), followed by HATU (32 mg, 0.084 mmol). After 15 min of stirring, dimethylamine (2 M in THF, 84 j L, 0.17 mmol) was added and the reaction was stirred for 1.5 h. After completion, saturated sodium bicarbonate (aq., 5 mL) was added and the mixture was extracted with CHCl (3 x 2 mL). The organic layers were combined and dried over NaSO, and concentrated in vacuo. The crude compound was purified using flash chromatography on SiO (gradient elution of CHCh with 2% NHMeOH = 0-20%) to afford (R)-2-(4-((6-((5-fluoro-4-(4-fluoro-1-isopropyl-2-methyl-1H-benzo[d]imidazol-6-yl)pyrimidin-2-yl)amino)pyridin-3-yl)methyl)-2-methylmorpholin-2-yl)-N,N-dimethylacetamide as a light orange oil. The oil was dissolved in water containing methanesulfonic acid (1.05 equiv.) and the solution was lyophilized to yield 7.2 mg of the mesylate salt as a tan solid.

Spectra of the mesylate salt (signals broadened by amide rotamers)

1H NMR (400 MHz, DMSO-d6 ) 8 10.42 (s, 1H), 8.70 (d, J = 3.6 Hz, 1H), 8.36 (s, 1H), 8.29 (d, J = 8.8 Hz, 1H), 8.23 (s, 1H), 7.86 (d, J = 8.9 Hz, 1H), 7.66 (d, J = 12.0 Hz, 1H), 4.80 (hept, J = 6.9 Hz, 1H), 4.31 (d, J = 22.5 Hz, 2H), 3.85 - 3.74 (m, 2H), 3.20 - 3.04 (m, 2H) 2.96 - 2.89 (m, 3H), 2.77 - 2.69 (m, 3H), 2.63 - 2.52 (m, 5H), 2.23 (s, 3H), 1.57 (d, J = 6.8 Hz, 6H), 1.36 (s, 2H), 1.28 - 1.06 (m, 3H). MS: [M+H]+ m/z 579.3.

Compound A56

To a cooled (0 °C) solution of (R)-2-(4-((6-((5-fluoro-4-(4-fluoro-1-isopropyl-2-methyl-1H-benzo[d]imidazol-6-yl)pyrimidin-2-yl)amino)pyridin-3-yl)methyl)-2-methylmorpholin-2-yl)acetic acid (31 mg, 0.056 mmol) in DMF (2.5 mL) was added N,N-diisopropylethylamine (50 µL, 0.28 mmol), followed by Ha Tu (32 mg, 0.084 mmol). After 15 min of stirring, 3,3-difluoroazetidine^HCl (22 mg, 0.17 mmol) was added and the reaction was stirred for 1.5 h. After completion, saturated sodium bicarbonate (aq., 5 mL) was added and the mixture was extracted with CHCl (3 x 2 mL). The organic layers were combined and dried over NaSO, and concentrated in vacuo. The crude compound was purified using flash chromatography on SO (gradient elution of CHCh with 2% NHMeOH = 0-20%) to afford (R)-1-(3,3-difluoroazetidin-1-yl)-2-(4-((6-((5-fluoro-4-(4-fluoro-1-isopropyl-2-methyl-1H-benzo[d]imidazol-6-yl)pyrimidin-2-yl)amino)pyridin-3-yl)methyl)-2-methylmorpholin-2-yl)ethan-1-one as a light orange oil. The oil was dissolved in water containing methanesulfonic acid (1.05 equiv.) and the solution was lyophilized to yield 7.2 mg of the mesylate salt as a tan solid.

Spectra of the mesylate salt (signals broadened by amide rotamers)

1H NMR (400 MHz, DMSO-da) 510.43 (s, 1H), 8.70 (d, J = 3.7 Hz, 1H), 8.36 (s, 1H), 8.28 (d, J = 8.8 Hz, 1H), 8.23 (d, J = 1.3 Hz, 1H), 7.92 - 7.82 (m, 1H), 7.72 - 7.62 (m, 1H), 4.80 (p, J = 6.9 Hz, 1H), 4.65 - 4.45 (m, 2H), 4.43 - 4.12 (m, 4H), 3.88 - 3.71 (m, 2H), 3.25 - 3.08 (m, 2H), 3.08 - 2.74 (m, 2H), 2.60 (s, 3H), 2.49 - 2.36 (m, 2H), 2.23 (s, 3H), 1.57 (d, J = 6.9 Hz, 6H), 1.17 (d, J = 6.1 Hz, 3H). MS: [M+H]+ m/z 627.3.

Compound A57

To a cooled (0 °C) solution of (R)-2-(4-((6-((5-fluoro-4-(4-fluoro-1-isopropyl-2-methyl-1H-benzo[d]imidazol-6-yl)pyrimidin-2-yl)amino)pyridin-3-yl)methyl)-2-methylmorpholin-2-yl)acetic acid (31 mg, 0.056 mmol) in DMF (2.5 mL) was added N,N-diisopropylethylamine (50 pL, 0.28 mmol), followed by HATU (32 mg, 0.084 mmol). After 15 min of stirring, diethylamine (18 pL, 0.17 mmol) was added and the reaction was stirred for 1 h. Upon completion, saturated sodium bicarbonate (aq., 5 mL) was added and the mixture was extracted with CHCl (3 x 2 mL). The organic layers were combined and dried over NaSO, and concentrated in vacuo. The crude compound was purified using flash chromatography on SiO (gradient elution of CHCl with 2% NHMeOH = 0-20%) to afford (R)-2-(4-((6-((5-fluoro-4-(4-fluoro-1-isopropyl-2-methyl-1H-benzo[d]imidazol-6-yl)pyrimidin-2-yl)amino)pyridin-3-yl)methyl)-2-methylmorpholin-2-yl)-N,N-diethylacetamide as a tan oil. The oil was dissolved in water containing methanesulfonic acid (1.05 equiv.) and the solution was lyophilized to yield 10.4 mg of the mesylate salt as a tan solid.

Spectra of the mesylate salt (signals broadened by amide rotamers)

1H NMR (400 MHz, DMSO-d6) 510.45 (s, 1H), 8.70 (d, J = 3.7 Hz, 1H), 8.37 (s, 1H), 8.33 - 8.18 (m, 2H), 7.93 - 7.82 (m, 1H), 7.67 (d, J = 11.9 Hz, 1H), 4.80 (p, J = 7.0 Hz, 1H), 4.44 -4.18 (m, 2H), 3.89 -3.70 (m, 2H), 3.68 - 3.58 (m, 2H), 3.23 - 3.09 (m, 4H), 3.06 - 2.77 (m, 2H), 2.60 (s, 3H), 2.56 - 2.43 (m, 2H), 2.23 (s, 3H), 1.57 (d, J = 6.9, 6H), 1.16 (d, J = 6.6 Hz, 3H), 1.06 - 0.98 (m, 3H), 0.96 - 0.86 (m, 3H). MS: [M+H]+ m/z 579.3.

Compound A58

To a cooled (0 °C) solution of (R)-2-(4-((6-((5-fluoro-4-(4-fluoro-1-isopropyl-2-methyl-1H-benzo[d]imidazol-6-yl)pyrimidin-2-yl)amino)pyridin-3-yl)methyl)-2-methylmorpholin-2-yl)acetic acid (31 mg, 0.056 mmol) in DMF (2.5 mL) was added N,N-diisopropylethylamine (50 pL, 0.28 mmol), followed by H<a>T<u>(32 mg, 0.084 mmol). After 15 min of stirring, azetidine^HCl (16, 0.17 mmol) was added and the reaction was stirred for 1 h. After completion, saturated sodium bicarbonate (aq. 5 mL) was added and the mixture was extracted with CHCl (3 x 2 mL). The organic layers were combined and dried over NaSO, and concentrated in vacuo. The crude compound was purified using flash chromatography on SiO (gradient elution of CHCl with 2% NH-MeOH = 0-20%) to afford (R)-1-(azetidin-1-yl)-2-(4-((6-((5-fluoro-4-(4-fluoro-1-isopropyl-2-methyl-1H-benzo[d]imidazol-6-yl)pyrimidin-2-yl)amino)pyridin-3-yl)methyl)-2-methylmorpholin-2-yl)ethan-1-one as a pale orange oil. The oil was dissolved in water containing methanesulfonic acid (1.05 equiv.) and the solution was lyophilized to yield 6.7 mg of the mesylate salt as a tan solid.

Spectra of the mesylate salt (signals broadened by amide rotamers)

1H NMR (400 MHz, DMSO-d6) 810.46 (s, 1H), 8.71 (d, J = 3.7 Hz, 1H), 8.43 -8.16 (m, 3H), 7.87 (d, J = 8.8 Hz, 1H), 7.68 (d, J = 12.0 Hz, 1H), 4.81 (p, J = 6.8 Hz, 1H), 4.45 -4.18 (m, 2H), 4.13 -4.01 (m, 2H), 3.86 -3.70 (m, 4H), 3.49 - 3.33 (m, 2H), 3.25 - 3.12 (m, 2H), 3.05 - 2.77 (m, 2H), 2.60 (s, 3H), 2.24 (s, 3H), 2.14 - 2.03 (m, 2H), 1.57 (d, J = 6.7 Hz, 6H), 1.16 (d, J = 6.0 Hz, 3H). MS: [M+H]+ m/z 591.3.

Compound A59

To a cooled (0 °C) solution of (R)-2-(4-((6-((5-fluoro-4-(4-fluoro-1-isopropyl-2-methyl-1H-benzo[d]imidazol-6-yl)pyrimidin-2-yl)amino)pyridin-3-yl)methyl)-2-methylmorpholin-2-yl)acetic acid (31 mg, 0.056 mmol) in DMF (2.5 mL) was added N,N-diisopropylethylamine (50 pL, 0.28 mmol), followed by HATU (32 mg, 0.084 mmol).

After 15 min of stirring, methylamine (2 M in MeOH, 84 pL, 0.17 mmol) was added and the reaction was stirred for 1 h. Upon completion, saturated sodium bicarbonate (aq., 5 mL) was added and the mixture was extracted with CHCl (3 x 2 mL). The organic layers were combined and dried over NaSO, and concentrated in vacuo. The crude compound was purified using flash chromatography on SiO2 (gradient elution of CH2Ch with 2% NH3 MeOH = 0-20%) to afford (R)-2-(4-((6-((5-fluoro-4-(4-fluoro-1-isopropyl-2-methyl-1H-benzo[d]imidazol-6-yl)pyrimidin-2-yl)amino)pyridin-3-yl)methyl)-2-methylmorpholin-2-yl)-N-methylacetamide as a light orange oil. The oil was dissolved in water containing methanesulfonic acid (1.05 equiv.) and the solution was lyophilized to yield 8.9 mg of the mesylate salt as a tan solid.

Spectra of the mesylate salt (signals broadened by amide rotamers)

1H NMR (400 MHz, DMSO-d6) 8 10.41 (s, 1H), 8.70 (d, J = 3.7 Hz, 1H), 8.36 (s, 1H), 8.29 (d, J = 8.5 Hz, 1H), 8.23 (d, J = 1.3 Hz, 1H), 7.86 (d, J = 9.2 Hz, 1H), 7.66 (dd, J = 11.9, 1.3 Hz, 1H), 4.80 (p, J = 6.9 Hz, 1H), 4.42 - 4.20 (m, 2H), 3.86 - 3.70 (m, 2H), 3.69 - 3.51 (m, 2H), 3.07 - 2.43 (m, 2H), 2.59 (s, 3H), 2.54 - 2.43 (m, 4H), 2.34 -2.19 (m, 6H), 1.57 (d, J = 6.8 Hz, 6H), 1.14 (d, J = 22.9 Hz, 3H). MS: [M+H]+ m/z 565.3.

Compound A60

To a cooled (0 °C) solution of (R)-2-(4-((6-((5-fluoro-4-(4-fluoro-1-isopropyl-2-methyl-1H-benzo[d]imidazol-6-yl)pyrimidin-2-yl)amino)pyridin-3-yl)methyl)-2-methylmorpholin-2-yl)acetic acid (31 mg, 0.056 mmol) in DMF (2.5 mL) was added N,N-diisopropylethylamine (50 pL, 0.28 mmol), followed by H<a>T<u>(32 mg, 0.084 mmol).

After 15 min of stirring, pyrrolidine (15 j L, 0.17 mmol) was added and the reaction was stirred for 1 h. Upon completion, saturated sodium bicarbonate (aq., 5 mL) was added and the mixture was extracted with CHCl3 (3 x 2 mL). The organic layers were combined and dried over Na2SO4, and concentrated in vacuo. The crude compound was purified using flash chromatography on SO2 (gradient elution of CH2Ch with 2% NH3 MeOH = 0-20%) to afford (R)-2-(4-((6-((5-fluoro-4-(4-fluoro-1-isopropyl-2-methyl-1H-benzo[d]imidazol-6-yl)pyrimidin-2-yl)amino)pyridin-3-yl)methyl)-2-methylmorpholin-2-yl)-1-(pyrrolidin-1-yl)ethan-1-one as a light orange oil. The oil was dissolved in water containing methanesulfonic acid (1.05 equiv.) and the solution was lyophilized to yield 7.9 mg of the mesylate salt as a tan solid.

Spectra of the mesylate salt (signals broadened by amide rotamers)

1H NMR (400 MHz, DMSO-da) 8 10.39 (s, 1H), 8.69 (d, J = 3.7 Hz, 1H), 8.36 (s, 1H), 8.29 (d, J = 8.6 Hz, 1H), 8.22 (d, J = 1.3 Hz, 1H), 7.85 (d, J = 8.9 Hz, 1H), 7.65 (d, J = 11.9 Hz, 1H), 4.79 (p, J = 6.9 Hz, 1H), 4.41 -4.20 (m, 2H), 3.87 - 3.72 (m, 2H), 3.69 - 3.55 (m, 2H), 3.23 - 3.08 (m, 4H), 3.04 - 2.76 (m, 2H), 2.59 (s, 3H), 2.52 -2.46 (m, 2H), 2.23 (s, 3H), 1.86 - 1.74 (m, 2H), 1.74 - 1.63 (m, 2H), 1.57 (d, J = 6.9 Hz, 6H), 1.17 (s, 3H). MS:

[M+H]+ m/z 605.3.

Example 2: Synthesis of compounds A43 and A44

A dry round-bottom flask was charged with 2,2-dimethylpiperazine (896 mg, 7.8 mmol) followed by dichloromethane (40 mL) and triethylamine (1.63 mL, 11.8 mmol). The reaction flask was cooled to -78 °C and benzyl chloroformate (1.23 mL, 8.6 mmol) was injected. The reaction was allowed to warm to room temperature over three hours. The solvent was removed in vacuo, and the reaction was purified using silica gel chromatography (0-10% methanol/dichloromethane). The major fraction was collected to yield, after solvent removal, benzyl 3,3-dimethylpiperazine-1-carboxylate (1.96 g, quant.). MS: [M+H]+ m/z 249.

Benzyl 3,3-dimethylpiperazine-1-carboxylate (3.77 g, 15.0 mmol) was dissolved in dichloromethane (75 mL) under nitrogen, and triethylamine (6.2 mL, 45.0 mmol) was injected, followed by di-tert-butyl dicarbonate (3.98 g, 18.2 mmol). After two hours, the conversion was incomplete based on TLC analysis, and a second portion of tert-butyl dicarbonate (2.00 g, 9.1 mmol) was added, and the reaction was stirred for 16 hours. The reaction was concentrated in vacuo and purified by silica gel chromatography (0-100% ethyl acetate/hexanes) to afford 4-benzyl 2,2-dimethylpiperazine-1,4-dicarboxylate 1 -(tert-butyl) as a clear oil (4.95 g, 95%). MS: [M+H]+ m/z 349.

A flask containing 4-benzyl 1-(tert-butyl) 2,2-dimethylpiperazine-1,4-dicarboxylate (4.95 g, 14.2 mmol) was charged with ethyl acetate (100 mL), followed by palladium on carbon (10%, 376 mg). Hydrogen gas was bubbled through the reaction mixture for 10 minutes, and stirring was continued under a hydrogen atmosphere for 5 hours. At this time, TLC analysis indicated that the reaction was complete (10% dichloromethane/methanol). The reaction was filtered through Celite, and the filtrate was concentrated in vacuo to yield tert-butyl 2,2-dimethylpiperazine-1-carboxylate (3.01 g, 99%) as a light yellow oil. MS: [M+H]+ m/z 215.

C o m p u e s t A 44

2,2-Dimethylpiperazine-1-carboxylate tert-butyl ester (352 mg, 1.6 mmol) was dissolved in dimethyl sulfoxide (16.5 mL) and acetic acid (0.45 mL). 6-((5-Fluoro-4-(4-fluoro-1-isopropyl-2-methyl-1H-benzo[d]imidazol-6-yl)pyrimidin-2-yl)amino)nicotinaldehyde (336 mg, 0.8 mmol) was added, and the reaction was stirred for 30 minutes, followed by the addition of sodium triacetoxyborohydride (694 mg, 3.3 mmol). The reaction was heated to 60 °C and continued for 17 hours before cooling to room temperature and pouring it into a mixture of chloroform and 1 N sodium hydroxide (100 mL portions). The organic phase was separated, dried over sodium sulfate, filtered, and concentrated to dryness. The crude product was dissolved in dichloromethane and trifluoroacetic acid (5 mL portions) and stirred for 2 hours. The reaction was concentrated in vacuo and dissolved in dimethyl sulfoxide. After filtration, the filtrate was purified by reverse-phase HPLC (10-60% acetonitrile/water, 0.25% trifluoroacetic acid for 15 minutes). The active fractions were pooled and concentrated on the Genevac to yield a white solid which was partitioned between ethyl acetate and saturated sodium bicarbonate to yield after concentration of the organic phase, N-(5-((3,3-dimethylpiperazin-1-yl)methyl)pyridin-2-yl)-5-fluoro-4-(4-fluoro-1-isopropyl-2-methyl-1H-benzo[d]imidazol-6-yl)pyrimidin-2-amine (241 mg, 58%) as a white solid. The mesylate salt was prepared by treating an acetonitrile solution of the compound with aqueous methanesulfonic acid and then freezing the solution and concentrating to a white solid in a lyophilizer. 1H NMR (500 MHz, Methanol-d4) 88.60 (d, J = 3.8 Hz, 1H), 8.40 (dd, J = 8.6, 0.8 Hz, 1H), 8.37 (d, J = 1.3 Hz, 1H), 8.28 (dd, J = 2.4, 0.8 Hz, 1H), 7.89 -7.82 (m, 2H), 5.01 -4.96 (m, 1H), 3.65 (s, 2H), 3.32 (t, J = 5.2 Hz, 2H), 2.76 (s, 5H), 2.52 (s, 2H), 1.78 (d, J = 6.9 Hz, 6H), 1.46 (s, 6H). MS: [M+H]+ m/z 507.

Compound A43

A flask containing the free base N-(5-((3,3-dimethylpiperazin-1-yl)methyl)pyridin-2-yl)-5-fluoro-4-(4-fluoro-1-isopropyl-2-methyl-1H-benzo[d]imidazol-6-yl)pyrimidin-2-amine (63 mg, 0.1 mmol) was dissolved in chloroform:methanol (9:1, 1 mL) and 37% formaldehyde (0.014 mL, 0.2 mmol) was added followed by sodium triacetoxyborohydride (87.3 mg, 0.4 mmol). After two hours, the entire reaction was poured into dichloromethane and sodium hydroxide (50 mL portions, 1 N). The organic phase was separated and concentrated in vacuo. The residue was purified using reverse phase HPLC (10-50% acetonitrile/water, 0.25% acetic acid over 15 min). The active fractions were pooled and concentrated in Genevac, and the residue was dissolved in acetonitrile and treated with methanesulfonic acid (1.05 equiv.). The resulting solution was frozen and concentrated in a lyophilizer to afford 5-fluoro-4-(4-fluoro-1-isopropyl-2-methyl-1H-benzo[d]imidazol-6-yl)-N-(5-((3,3,4-trimethylpiperazin-1-yl)methyl)pyridin-2-yl)pyrimidin-2-amine (23 mg, 42%) as the corresponding mesylate salt.

1H NMR (500 MHz, M e tano l^) 88.68 (d, J = 3.7 Hz, 1H), 8.38 (d, J = 1.3 Hz, 1H), 8.31 (d, J = 2.1 Hz, 1H), 8.25 - 8.15 (s, 1H), 8.00 (s, 1H), 7.93 - 7.84 (m, 1H), 4.99 (q, J = 6.9 Hz, 1H), 3.68 (q, J = 13.5 Hz, 2H), 3.50 -3.40 (m, 2H), 3.15 (d, J = 12.7 Hz, 1H), 2.92 (t, J = 14.0 Hz, 1H), 2.87 (s, 3H), 2.76 (s, 3H), 2.75 (s, 3H), 2.51 (s, 1H), 2.35 (d, J = 12.8 Hz, 1H), 1.78 (d, J = 6.9 Hz, 6H), 1.51 (s, 3H), 1.44 (s, 3H). MS: [M+H]+ m/z 521.

Example 3: Synthesis of compounds A30, A31, A34, A37, A39 and A41

A solution of tert-butyl-8-oxo-6-oxa-2,9-diazaspiro[4.5]decane-2-carboxylate (450 mg) in methanol was purified by SFC. Separation returned (S)-tert-butyl 8-oxo-6-oxa-2,9-diazaspiro[4.5]decane-2-carboxylate (160.5 mg) and (R)-tert-butyl 8-oxo-6-oxa-2,9-diazaspiro[4.5]decane-2-carboxylate (209.3 mg) as white solids. The following reactions were carried out on both enantiomers, but the yield and exact procedure are reported from the first peak.

A round-bottom flask was charged with (S)-8-oxo-6-oxa-2,9-diazaspiro[4.5]decane-2-carboxylic acid tert-butyl ester (160.5 mg, 0.63 mmol) and tetrahydrofuran was injected to produce a clear solution (4 mL). Borane in tetrahydrofuran was injected under a nitrogen atmosphere (1 M, 1.9 mL, 1.9 mmol), and the reaction was stirred at room temperature for three hours. The solvent was removed under reduced pressure, and the residue was dissolved in methanol (5 mL). Palladium on charcoal (10%, 10 mg) was added to the resulting solution, and the suspension was stirred for one hour and filtered through Celite. The filter cake was washed with dichloromethane. Concentration in vacuo gave (S)-6-oxa-2,9-diazaspiro[4.5]decane-2-carboxylic acid tert-butyl ester (112 mg, 73%). MS: [M+H]+ m/z 243.

Compound A41

A round-bottom flask was charged with (S)-6-oxa-2,9-diazaspiro[4.5]decane-2-carboxylic acid tert-butyl ester (112 mg, 0.45 mmol) and 6-((5-fluoro-4-(4-fluoro-1-isopropyl-2-methyl-1H-benzo[d]imidazol-6-yl)pyrimidin-2-yl)amino)nicotinaldehyde (226 mg, 0.55 mmol). Dimethyl sulfoxide (10 mL) and acetic acid (1 mL) were injected under nitrogen and the reaction was heated at 60 °C for a period of 18 hours. The reaction was allowed to cool and diluted with sodium hydroxide (1 N aq., 20 mL) and water (100 mL). A cloudy white precipitate formed which was stirred for one hour and then filtered. The white solid was washed with several portions of water (5 mL each) and dried in a lyophilizer to yield (S)-9-((6-((5-fluoro-4-(4-fluoro-1-isopropyl-2-methyl-1H-benzo[d]imidazol-6-yl)pyrimidin-2-yl)amino)pyridin-3-yl)methyl)-6-oxa-2,9-diazaspiro[4.5]decane-2-carboxylic acid tert-butyl ester (265 mg, 90%).

1H NMR (500 MHz, DMSO-d6) 88.71 (d, J = 3.7 Hz, 1H), 8.31 (s, 1H), 8.26 (d, J = 8.5 Hz, 1H), 8.21 (d, J = 2.2 Hz, 1H), 7.73 -7.67 (m, 2H), 4.86 (p, J = 7.0 Hz, 1H), 3.74 -3.58 (m, 4H), 3.32 -3.11 (m, 4H), 2.66 (s, 3H), 2.50 - 2.22 (m, 4H), 1.99 (d, J = 11.8 Hz, 1H), 1.82 (s, 1H), 1.65 (dd, J = 7.0, 3.1 Hz, 6H), 1.38 (s, 9H). MS: [M+H]+ m/z 635.

Compound A34

(S)-9-((6-((5-fluoro-4-(4-fluoro-1-isopropyl-2-methyl-1H-benzo[d]imidazol-6-yl)pyrimidin-2-yl)amino)pyridin-3-yl)methyl)-6-oxa-2,9-diazaspiro[4.5]decane-2-carboxylic acid tert-butyl ester (248 mg, 0.46 mmol) was dissolved in chloroform (6 mL) and trifluoroacetic acid was added via pipette (3 mL). The resulting orange solution was stirred for thirty minutes and concentrated to yield (R)-N-(5-((6-oxa-2,9-diazaspiro[4.5]decan-9-yl)methyl)pyridin-2-yl)-5-fluoro-4-(4-fluoro-1-isopropyl-2-methyl-1H-benzo[d]imidazol-6-yl)pyrimidin-2-amine as a trifluoroacetate salt which was used without further purification (301 mg, quant.).

A portion of this sample was liberated with aqueous sodium bicarbonate and dichloromethane and purified by preparative HPLC (10-35% acetonitrile/water/0.25% acetic acid for 15 minutes). This yielded an analytical sample (3.9 mg) that was used for NMR analysis and in biochemical and cell-based assays.

1H NMR (500 MHz, DMSO-da) 88.70 (d, J = 3.7 Hz, 1H), 8.30 (d, J = 1.3 Hz, 1H), 8.28 - 8.18 (m, 2H), 7.75 -7.64 (m, 2H), 4.86 (p, J = 6.9 Hz, 1H), 3.58 - 3.55 (m, 2H), 3.46 (m, 2H), 2.77 (d, J = 11.2 Hz, 2H), 2.66 - 2.60 (m, 5H), 2.42 - 2.33 (m, 2H), 2.28 (d, J = 12.8 Hz, 1H), 1.76 (s, 1H), 1.68 - 1.59 (m, 6H). MS: [M+H]+

m/z 535.

Compound A39

(R)-N-(5-((6-oxa-2,9-diazaspiro[4.5]decan-9-yl)methyl)pyridin-2-yl)-5-fluoro-4-(4-fluoro-1-isopropyl-2-methyl-1H-benzo[d]imidazol-6-yl)pyrimidin-2-amine 2,2,2-trifluoroacetate (32 mg, 0.049 mmol) was dissolved in dimethylformamide (3 mL) and treated with formaldehyde (0.10 mL, 37% in water, 1.68 mmol) followed by sodium triacetoxyborohydride (32 mg, 0.15 mmol). The reaction was stirred for three hours and filtered through celite. The filtrate was purified directly by HPLC (10-35% acetonitrile/water/0.25% acetic acid for 15 minutes). The active fractions were pooled and concentrated in a lyophilizer to yield (R)-5-fluoro-4-(4-fluoro-1-isopropyl-2-methyl-1H-benzo[d]imidazol-6-yl)-N-(5-((2-methyl-6-oxa-2,9-diazaspiro[4.5]decan-9-yl)methyl)pyridin-2-yl)pyrimidin-2-amine (4.7 mg, 18%) as a white solid.

1H NMR (500 MHz, DMSO-d6) 88.71 (d, J = 3.7 Hz, 1H), 8.31 (d, J = 1.2 Hz, 1H), 8.27 - 8.21 (m, 2H), 7.74 -7.67 (m, 2H), 4.87 (p, J = 6.9 Hz, 1H), 3.81 -3.48 (m, 4H), 2.70 -2.65 (m, 5H), 2.50 -2.27 (m, 5H), 2.17 (s, 3H), 1.74 (q, J = 7.0, 6.3 Hz, 1H), 1.65 (d, J = 6.8 Hz, 6H). MS: [M+H]+ m/z 549.

Compound A37

(R)-N-(5-((6-oxa-2,9-diazaspiro[4.5]decan-9-yl)methyl)pyridin-2-yl)-5-fluoro-4-(4-fluoro-1-isopropyl-2-methyl-1H-benzo[d]imidazol-6-yl)pyrimidin-2-amine 2,2,2-trifluoroacetate (32 mg, 0.049 mmol) was dissolved in dimethylformamide (3 mL) and treated with triethylamine (0.05 mL, 0.36 mmol) and acetyl chloride (10 pL, 0.18 mmol). The reaction was stirred for three hours and quenched with several drops of methanol. After filtration through Celite, the filtrate was purified by reverse phase HPLC (10-60% acetonitrile/water/0.25% acetic acid over 15 min). The active fractions were pooled and concentrated in a lyophilizer to yield (S)-1-(9-((6-((5-fluoro-4-(4-fluoro-1-isopropyl-2-methyl-1H-benzo[d]imidazol-6-yl)pyrimidin-2-yl)amino)pyridin-3-yl)methyl)-6-oxa-2,9-diazaspiro[4.5]decan-2-yl)ethan-1-one (4.0 mg, 14%) as a white solid.

1H NMR (500 MHz, DMSO-d6) 88.71 (d, J = 3.7 Hz, 1H), 8.33 - 8.28 (m, 1H), 8.28 - 8.20 (m, 2H), 7.76 - 7.65 (m, 2H), 4.86 (p, J = 6.9 Hz, 1H), 3.76 -3.50 (m, 4H), 2.66 (s, 3H), 2.50 -2.25 (m, 4H), 2.14 -1.95 (m, 2H), 1.94 - 1.90 (m, 3H), 1.67 - 1.63 (m, 6H). MS: [M+H]+ m/z 577.

C o m p u e s t A31

(R)-N-(5-((6-oxa-2,9-diazaspiro[4.5]decan-9-yl)methyl)pyridin-2-yl)-5-fluoro-4-(4-fluoro-1-isopropyl-2-methyl-1H-benzo[d]imidazol-6-yl)pyrimidin-2-amine 2,2,2-trifluoroacetate (32 mg, 0.049 mmol) was dissolved in dimethylformamide (3 mL) and treated with triethylamine (0.05 mL, 0.36 mmol) and mesyl chloride (10 L). The reaction was stirred for three hours and quenched with several drops of methanol. After filtration through Celite, the filtrate was purified using reverse-phase HPLC (10-60% acetonitrile/water/0.25% acetic acid for 15 minutes). The active fractions were pooled and concentrated in a lyophilizer to yield (S)-5-fluoro-4-(4-fluoro-1-isopropyl-2-methyl-1H-benzo[d]imidazol-6-yl)-N-(5-((2-(methylsulfonyl)-6-oxa-2,9-diazaspiro[4.5]decan-9-yl)methyl)pyridin-2-yl)pyrimidin-2-amine (3.8 mg, 13%) as a white solid.

1H NMR (500 MHz, DMSO-da) 88.71 (d, J = 3.8 Hz, 1H), 8.31 (d, J = 1.2 Hz, 1H), 8.28 - 8.17 (m, 2H), 7.76 -7.67 (m, 2H), 4.86 (p, J = 6.9 Hz, 1H), 3.69 (t, J = 4.8 Hz, 2H), 3.50 - 3.45 (m, 3H), 3.28 (dd, J = 8.8, 5.3 Hz, 2H), 3.23 - 3.14 (m, 1H), 2.85 (s, 3H), 2.66 (s, 3H), 2.50 - 2.46 (m, 2H), 2.31 (d, J = 11.4 Hz, 2H), 2.09 - 1.96 (m, 1H), 1.87 (dt, J = 13.1, 8.8 Hz, 1H), 1.65 (d, J = 6.8 Hz, 6H). MS: [M+H]+ m/z 613.

Compound A30

(R)-N-(5-((6-oxa-2,9-diazaspiro[4.5]decan-9-yl)methyl)pyridin-2-yl)-5-fluoro-4-(4-fluoro-1-isopropyl-2-methyl-1H-benzo[d]imidazol-6-yl)pyrimidin-2-amine 2,2,2-trifluoroacetate (32 mg, 0.049 mmol) was dissolved in dimethylformamide (3 mL) and treated with triethylamine (0.05 mL, 0.36 mmol) and methyl chloroformate (10 L). The reaction was stirred for three hours and quenched with several drops of methanol. After filtration through Celite, the filtrate was purified by reverse-phase HPLC (10-60% acetonitrile/water/0.25% acetic acid for 15 minutes). The active fractions were pooled and concentrated in the lyophilizer to yield (S)-9-((6-((5-fluoro-4-(4-fluoro-1-isopropyl-2-methyl-1H-benzo[d]imidazol-6-yl)pyrimidin-2-yl)amino)pyridin-3-yl)methyl)-6-oxa-2,9-diazaspiro[4.5]decane-2-carboxylic acid methyl ester (5.0 mg, 18%) as a white solid. 1H NMR (500 MHz, DMSO-d6) 88.71 (d, J = 3.7 Hz, 1H), 8.35 -8.16 (m, 3H), 7.78 -7.62 (m, 2H), 4.86 (p, J = 6.8 Hz, 1H), 3.73 - 3.60 (m, 2H), 3.57 (s, 3H), 3.48 (s, 2H), 3.37 - 3.11 (m, 4H), 2.66 (s, 3H), 2.49 - 2.22 (m, 4H), 2.11 -1.77 (m, 2H), 1.65 (d, J = 6.9 Hz, 6H). MS: [M+H]+ m/z 593.

Example 4: Synthesis of compounds A23 and A28

To a cooled solution of (S)-2-allyl-2-methylmorpholine (6.74 g, 47.8 mmol) in dichloromethane (120 mL, 0.4 M) under nitrogen was added triethylamine (9.80 mL, 71.7 mmol), and the mixture was cooled in an ice/water bath. 4-Toluenesulfonyl chloride (10.03 g, 52.3 mmol) was added in several portions, the reaction was stirred for 30 minutes, and the reaction was allowed to warm to room temperature over a period of 90 minutes. Excess reagent was quenched by the addition of water (50 mL) and saturated sodium bicarbonate (50 mL). The mixture was transferred to a separatory funnel. The organic phase was separated, and the aqueous phase was washed with two additional portions of dichloromethane (30 mL). The combined organic phases were concentrated onto silica gel and the residue was loaded dry onto a silica flash column (hexanes/dichloromethane/2% ethyl acetate gradient: 0.100%). The active fractions were pooled and concentrated to afford (S)-2-allyl-2-methyl-4-tosylmorpholine (11.95 g, 85%) as an off-white solid.<m>S: [M+H]+ m/z 296.

A flask containing (S)-2-allyl-2-methyl-4-tosylmorpholine (11.95 g, 40.48 mmol) was charged with tert-butyl alcohol, methanol, and water (125 mL: 75 mL: 2 mL) and purged with nitrogen. To the resulting solution were added 1,4-benzoquinone (6.56 g, 60.72 mmol) and PdCh(CH3CN)2 (313 mg, 1.21 mmol). The reaction was gently heated to 50 °C and allowed to cool after three hours. The resulting solution was quenched with 1 N hydrochloric acid (15 mL) and diluted after 30 minutes with ethyl acetate (300 mL). The aqueous phase was separated and the remaining organic portion was washed several times with sodium hydroxide solution (1 N, 50 mL portions) and saturated sodium bicarbonate (50 mL). The organic portion was concentrated onto silica gel and loaded dry onto a silica flash column (hexanes/ethyl acetate gradient: 0-100%). The active fractions were pooled and concentrated to yield (S)-1-(2-methyl-4-tosylmorpholin-2-yl)propan-2-one (10.55 g, 85%) as an off-white solid. MS: [M+H]+ m/z 312.

A glass bottle installed in a bomb reactor was charged with palladium on carbon (10%, 4.11 g) and gently purged with nitrogen gas to cover the catalyst bed. Under nitrogen, tetrahydrofuran (50 mL) was added via pipette, and the mixture was stirred vigorously. Dimethylamine in tetrahydrofuran (100 mL, 2 M) was mixed with acetic acid (5.77 mL, 100 mmol), and the resulting solution was transferred to the catalyst solution via pipette. Finally, (S)-1-(2-methyl-4-tosylmorpholin-2-yl)propan-2-one (8.23 g, 26.4 mmol) was dissolved in tetrahydrofuran (50 mL) and transferred to the catalyst solution. The bomb reactor was sealed under a nitrogen atmosphere and connected to a high-pressure hydrogen tank. The reactor was pressurized to 1000 psi and the pressure was released three times. The pressurized reaction was then sealed and heated to 60 °C for a period of 38 hours. The reaction was allowed to cool and excess hydrogen gas was released. The catalyst solution was filtered through Celite and the cake was washed with ethyl acetate without allowing the palladium to dry. Caution: The remaining palladium catalyst can be pyrophoric and was kept wet with ethyl acetate and then water before being transferred to a waste container. After filtration, the filtrate was diluted with ethyl acetate (300 mL) and transferred to a separatory funnel. The organic phase was washed with sodium hydroxide solution (2 N, 200 mL) and saturated sodium bicarbonate. The combined organic washings were concentrated on silica gel and the residue was loaded dry onto a silica flash column (dichloromethane/methanol gradient: 0-25%). Three fractions were collected, the first containing the starting material, followed by (S)-N,N-dimethyl-1-((R)-2-methyl-4-tosylmorpholin-2-yl)propan-2-amine (2.33 g, 26%) and finally by the product (S)-N,N-dimethyl-1-((S)-2-methyl-4-tosylmorpholin-2-yl)propan-2-amine (3.25 g, 36%). MS: [M+H]+ m/z 341.

A round-bottom flask was charged with (S)-N,N-dimethyl-1-((S)-2-methyl-4-tosylmorpholin-2-yl)propan-2-amine (2.84 g, 8.35 mmol). Tetrahydrofuran (42 mL) was injected under nitrogen, and the resulting solution was cooled to -78 °C. Lithium aluminum hydride (2.14 g, 56.36 mmol) was added in a single portion, and the reaction was allowed to warm to room temperature. The reaction was heated at 60 °C for a period of 50.5 hours. At this time, the reaction was cooled again in a dry ice/acetone bath, and sodium hydroxide (1 N, 2.5 mL) and water (2.5 mL) were added dropwise with vigorous evolution of hydrogen gas. After heating for thirty minutes, magnesium sulfate (20.0 g) was added and the reaction was diluted with one volume of diethyl ether and stirred vigorously. After 30 minutes, the mixture was filtered through Celite and the filter cake was washed with dichloromethane. The filtrate was concentrated to afford (S)-N,N-dimethyl-1-((S)-2-methylmorpholin-2-yl)propan-2-amine (1.51 g, 97% crude) as a pure yellow oil. MS: [M+H]+ m/z 187.

C o m p u e s t A 23

(S)-N,N-Dimethyl-1-((S)-2-methylmorpholin-2-yl)propan-2-amine (1.51 g, 8.35 mmol) was transferred to a round-bottom flask and dissolved in DMSO (80 mL) and acetic acid (4.58 mL, 79.5 mmol). 6-((5-Fluoro-4-(4-fluoro-1-isopropyl-2-methyl-1H-benzo[d]imidazol-6-yl)pyrimidin-2-yl)amino)nicotinaldehyde (3.24 g, 7.95 mmol) was added to give a milky-white suspension. After 30 min, sodium triacetoxyborohydride (5.05 g, 23.85 mmol) was added and the reaction was heated at 60 °C for 14.5 h. After cooling, the resulting clear yellow solution was poured into sodium hydroxide (0.5 N, 1 L) and the aqueous phase was washed with three portions of chloroform (600 mL each). The combined organic washings were dried over sodium sulfate, filtered, and concentrated onto silica gel. The residue was loaded dry onto a silica flash column (dichloromethane/methanol gradient with 17 N methanol/3% ammonia: 0-50% - followed by switching the strong phase to methanol/4% ammonium hydroxide). The active fractions were pooled and concentrated to yield N-(5-(((S)-2-((S)-2-(dimethylamino)propyl)-2-methylmorpholino)methyl)pyridin-2-yl)-5-fluoro-4-(4-fluoro-1-isopropyl-2-methyl-1H-benzo[d]imidazol-6-yl)pyrimidin-2-amine (3.30 g, 67%). The free base was analyzed by proton NMR and LC/MS indicating the desired product. The entire portion was dissolved in acetonitrile and aqueous methanesulfonic acid (0.23 M, 26 mL) was added. The saline solution was frozen in a dry ice acetone bath and concentrated in the lyophilizer for several days.

1H NMR (500 MHz, Chloroform-d) 88.43 (d, J = 3.7 Hz, 1H), 8.39 (dd, J = 8.5, 0.8 Hz, 1H), 8.25 (dd, J = 2.3, 0.8 Hz, 1H), 8.22 (s, 1H), 8.19 (d, J = 1.3 Hz, 1H), 7.79 (dd, J = 11.5, 1.3 Hz, 1H), 7.69 (dd, J = 8.6, 2.3 Hz, 1H), 4.74 (p, J = 7.0 Hz, 1H), 3.76 (t, J = 4.9 Hz, 2H), 3.42 (s, 2H), 2.90 - 2.62 (m, 4H), 2.47 - 2.39 (m, 1H), 2.36 (q, J = 5.3 Hz, 1H), 2.33 -2.25 (m, 1H), 2.25 -2.18 (m, 1H), 2.18 (s, 6H), 1.90 (dd, J = 14.1, 3.8 Hz, 1H), 1.72 (d, J = 7.0 Hz, 6H), 1.39 (dd, J = 14.1,6.9 Hz, 1H), 1.27 (s, 3H), 0.99 (d, J = 6.5 Hz, 3H). MS: [M+H]+ m/z 579.

Compound A28

(S)-N,N-dimethyl-1-((R)-2-methyl-4-tosylmorpholin-2-yl)propan-2-amine participated in the final two-step sequence in an identical manner to provide N-(5-(((S)-2-((R)-2-(dimethylamino)propyl)-2-methylmorpholino)methyl)pyridin-2-yl)-5-fluoro-4-(4-fluoro-1-isopropyl-2-methyl-1H-benzo[d]imidazol-6-yl)pyrimidin-2-amine methanesulfonate.

1H NMR (500 MHz, Chloroform-d) 88.44 (d, J = 3.8 Hz, 1H), 8.40 (dd, J = 8.5, 0.8 Hz, 1H), 8.30 (s, 1H), 8.25 (dd, J = 2.3, 0.8 Hz, 1H), 8.19 (d, J = 1.3 Hz, 1H), 7.82 - 7.77 (m, 1H), 7.69 (dd, J = 8.6, 2.3 Hz, 1H), 4.73 (td, J = 14.0, 7.1 Hz, 1H), 3.71 (t, J = 4.9 Hz, 2H), 3.51 - 3.31 (m, 2H), 2.83 (q, J = 6.3, 5.7 Hz, 1H), 2.69 (s, 3H), 2.38 - 2.34 (m, 2H), 2.24 (d, J = 11.1 Hz, 1H), 2.19 (s, 6H), 1.71 (d, J = 7.0 Hz, 6H), 1.60 (dd, J = 14.2, 7.1 Hz, 1H), 1.25 (s, 3H), 1.02 (d, J = 6.6 Hz, 3H). MS: [M+H]+ m/z 579.

Example 5: Synthesis of compounds A61-A64

C o m p u e s t A61

To a solution of 6-oxa-1-azaspiro[3.3]heptane hemioxalate (22 mg, 0.073 mmol) and 6-((5-fluoro-4-(4-fluoro-1-isopropyl-2-methyl-1H-benzo[d]imidazol-6-yl)pyrimidin-2-yl)amino)nicotinaldehyde (50 mg, 0.12 mmol) in DMSO (1.5 mL) was added acetic acid (70 j L, 1.2 mmol), followed by sodium triacetoxyborohydride (52 mg, 0.25 mmol). The reaction was heated to 60 °C and stirred for 24 h. After completion, the mixture was cooled to room temperature and sodium hydroxide solution (1 M aq., 2 mL) was added, followed by water (2 mL). The mixture was extracted with CHCl (3 x 3 mL). The organic layers were combined and dried over NaSO, and concentrated in vacuo. The crude compound was purified using flash chromatography on SiO (gradient elution of CHCl with 2% NHMeOH = 0-20%) to afford N-(5-((6-oxa-1-azaspiro[3.3]heptan-1-yl)methyl)pyridin-2-yl)-5-fluoro-4-(4-fluoro-1-isopropyl-2-methyl-1H-benzo[d]imidazol-6-yl)pyrimidin-2-amine as a clear oil. The oil was dissolved in water containing methanesulfonic acid (1.05 equiv.) and the solution was lyophilized to yield 42 mg of the mesylate salt as a white solid.

Mesylate salt spectra

1H NMR (400 MHz, DMSO-da) 810.35 (s, 1H), 10.28 (bs, 1H), 8.68 (d, J = 3.7 Hz, 1H), 8.38 (s, 1H), 8.27 (d, J = 8.7 Hz, 1H), 8.22 (d, J = 1.3 Hz, 1H), 7.86 (dd, J = 8.8, 2.4 Hz, 1H), 7.65 (dd, J = 11.9, 1.2 Hz, 1H), 5.28 (d, J = 9.3 Hz, 1H), 4.93 -4.58 (m, 5H), 4.39 -4.27 (m, 1H), 3.96 -3.81 (m, 1H), 3.68 - 3.54 (m, 1H), 2.70 -2.55 (m, 5H), 2.23 (s, 3H), 1.57 (d, J = 6.9 Hz, 6H). MS: [M+H]+ m/z 492.2.

Compound A62

To a solution of 2-thia-6-azaspiro[3.3]heptane-2,2-dioxide hydrochloride (27 mg, 0.15 mmol) and 6-((5-fluoro-4-(4-fluoro-1-isopropyl-2-methyl-1H-benzo[d]imidazol-6-yl)pyrimidin-2-yl)amino)nicotinaldehyde (50 mg, 0.12 mmol) in DMSO (1.5 mL) was added acetic acid (70 j L, 1.2 mmol), followed by sodium triacetoxyborohydride (52 mg, 0.25 mmol). The reaction was heated to 60 °C and stirred for 24 h. After completion, the mixture was cooled to room temperature and aqueous sodium hydroxide solution (1 M, 2 mL) was added, followed by water (2 mL). The mixture was extracted with CHCl (3 x 3 mL). The organic layers were combined and dried over NaSO, and concentrated in vacuo. The crude compound was purified using flash chromatography on SO (gradient elution of CHCl with 2% NHMeOH = 0-20%) to afford 6-((6-((5-fluoro-4-(4-fluoro-1-isopropyl-2-methyl-1H-benzo[d]imidazol-6-yl)pyrimidin-2-yl)amino)pyridin-3-yl)methyl)-2-thia-6-azaspiro[3.3]heptane 2,2-dioxide as a clear oil. The oil was dissolved in water containing methanesulfonic acid (1.05 equiv.) and the solution was lyophilized to yield 8.5 mg of the mesylate salt as a white solid.

Mesylate salt spectra

1H NMR (400 MHz, DMSO-da) 510.36 (s, 1H), 10.13 (bs, 1H), 8.68 (d, J = 3.7 Hz, 1H), 8.32 (d, J = 2.4 Hz, 1H), 8.26 (d, J = 8.7 Hz, 1H), 8.22 (d, J = 1.3 Hz, 1H), 7.78 (dd, J = 8.7, 2.4 Hz, 1H), 7.65 (dd, J = 11.9, 1.3 Hz, 1H), 4.79 (p, J = 6.9 Hz, 1H), 4.53 -4.37 (m, 6H), 4.31 (d, J = 5.6 Hz, 2H), 4.27 -4.16 (m, 2H), 2.59 (s, 3H), 2.23 (s, 3H), 1.57 (d, J = 6.9 Hz, 6H). MS: [M+H]+ m/z 540.2.

Compound A63

To a solution of 2-azaspiro[3.3]heptane hemioxalate (21 mg, 0.075 mmol) and 6-((5-fluoro-4-(4-fluoro-1-isopropyl-2-methyl-1H-benzo[d]imidazol-6-yl)pyrimidin-2-yl)amino)nicotinaldehyde (50 mg, 0.12 mmol) in DMSO (1.5 mL) was added acetic acid (70 pL, 1.2 mmol), followed by sodium triacetoxyborohydride (52 mg, 0.25 mmol). The reaction was heated to 60 °C and stirred for 24 h. Upon completion, the mixture was cooled to room temperature and sodium hydroxide solution (1 M aq, 2 mL) was added, followed by water (2 mL). The mixture was extracted with CHCl (3 x 3 mL). The organic layers were combined and dried over Na2SO4, and concentrated in vacuo. The crude compound was purified using flash chromatography on SiO2 (gradient elution of CH2Ch with 2% NH3 MeOH = 0-20%) to afford N-(5-((2-azaspiro[3.3]heptan-2-yl)methyl)pyridin-2-yl)-5-fluoro-4-(4-fluoro-1-isopropyl-2-methyl-1H-benzo[d]imidazol-6-yl)pyrimidin-2-amine as a clear oil. The oil was dissolved in water containing methanesulfonic acid (1.05 equiv.) and the solution was lyophilized to yield 42 mg of the mesylate salt as a white solid.

Mesylate salt spectra

1H NMR (400 MHz, DMSO-d6 ) 510.35 (s, 1H), 9.85 (bs, 1H), 8.68 (d, J = 3.7 Hz, 1H), 8.31 (d, J = 2.3 Hz, 1H), 8.28 - 8.17 (m, 2H), 7.79 (dd, J = 8.7, 2.4 Hz, 1H), 7.65 (dd, J = 11.9, 1.3 Hz, 1H), 4.80 (p, J = 6.9 Hz, 1H), 4.23 (d, J = 6.0 Hz, 2H), 4.12 - 4.00 (m, 2H), 4.00 - 3.90 (m, 2H), 2.59 (s, 3H), 2.23 (s, 3H), 2.15 (dd, J = 8.8, 6.6 Hz, 2H), 2.12 - 2.04 (m, 2H), 1.76 - 1.63 (m, 2H), 1.57 (d, J = 6.9 Hz, 6H). MS: [M+H]+ m/z 490.3.

Compound A64

To a solution of 6,6-difluoro-2-aza-spiro[3.3]heptane trifluoroacetate (37 mg, 0.15 mmol) and 6-((5-fluoro-4-(4-fluoro-1-isopropyl-2-methyl-1H-benzo[d]imidazol-6-yl)pyrimidin-2-yl)amino)nicotinaldehyde (50 mg, 0.12 mmol) in DMSO (1.5 mL) was added acetic acid (70 pL, 1.2 mmol), followed by sodium triacetoxyborohydride (52 mg, 0.25 mmol). The reaction was heated to 60 °C and stirred for 24 h. Upon completion, the mixture was cooled to room temperature, and aqueous sodium hydroxide solution (1 M, 2 mL) was added, followed by water (2 mL). The mixture was extracted with CHCl (3 x 3 mL). The organic layers were combined and dried over NaSO, and concentrated in vacuo. The crude compound was purified using flash chromatography on SiO (gradient elution of CHCl with 2% NHMeOH = 0-20%) to afford N-(5-((6,6-difluoro-2-azaspiro[3.3]heptan-2-yl)methyl)pyridin-2-yl)-5-fluoro-4-(4-fluoro-1-isopropyl-2-methyl-1H-benzo[d]imidazol-6-yl)pyrimidin-2-amine as a clear oil. The oil was dissolved in water containing methanesulfonic acid (1.05 equiv.) and the solution was lyophilized to yield 45 mg of the mesylate salt as a white solid.

Mesylate salt spectra

1H NMR (400 MHz, DMSO-da) 810.36 (s, 1H), 9.99 (bs, 1H), 8.68 (d, J = 3.7 Hz, 1H), 8.32 (d, J = 2.3 Hz, 1H), 8.28 - 8.19 (m, 2H), 7.79 (dd, J = 8.7, 2.4 Hz, 1H), 7.65 (dd, J = 11.9, 1.3 Hz, 1H), 4.80 (p, J = 6.9 Hz, 1H), 4.32 -4.21 (m, 4H), 4.14 -4.02 (m, 2H), 2.92 -2.77 (m, 4H), 2.59 (s, 3H), 2.23 (s, 3H), 1.57 (d, J = 6.9 Hz, 6H). MS:

[M+H]+ m/z 492.2

Biological experiments

General methods

Cell line growth retardation assay

Cells were seeded at densities of 1,000-5,000 cells per well in 48-well tissue culture plates. After a 24-hour rest period, cells were treated with compound at 10 μM, 2 μM, 0.4 μM, 0.08 μM, 0.016 μM, and 0.0032 μM. A group of cells was treated with the vehicle in which the compound was prepared and served as a control. Cells were cultured in the presence of compounds for 6 days and counted on day 0 and day 6. All cell counting was performed using a Synentec Cellavista plate imager. Cells that did not receive compound were counted on day 1, and this count was used as a reference value for calculating growth inhibition. Growth inhibition was calculated as a ratio of the doubling of the cell population in the presence of compound to that in the absence of compound. If treatment resulted in a net loss of cells relative to baseline, percent lethality was defined as the decrease in cell numbers in treated wells compared to counts on day 1 of untreated wells after seeding. IC50 values for each compound (see Table 3; reported in M) were calculated by curve fitting to the data points from each dose-response assay using the Proc NLIN function in SAS for Windows version 9.2 (SAS Institute, Inc.).

Cdk4 and 6 enzyme inhibition assay

For the Ki determination assay, 200 jiM stock solutions of compounds were subjected to serial half-log dilution using 100% DMSO as the solvent. Ten different concentrations were prepared, with a dilution endpoint of 6 x 10.9 M in 100% DMSO. 100% DMSO was used as a control. Aliquots of each serial dilution were made into separate wells of a 96-well plate, and 90 jiL of water was added to each well. The plate was shaken thoroughly, and 5 jiL from each well of the plate was transferred to the wells of the assay plate. The final volume of the wells in the assay plate was 50 jiL. All compounds were tested at 10 assay concentrations ranging from 2 x 10.6 M to 6 x 10.11 M. The final DMSO concentration in the assay plate wells was 1% in all cases. The Ki for the compounds are presented in Table 3 in nM.

A radiometric protein kinase assay (PanQinase® Activity Assay) was used to measure the kinase activity of six protein kinases. All kinase assays were performed in 96-well FlashPlates™ from PerkinElmer (Boston, MA, USA). The assay for all protein kinases contained 70 mM HEpES-NaOH pH 7.5, 3 mM MgCh2, 3 mM MnCh2, 3<ji>M Na orthovanadate, 1.2 mM DTT, 50 µg/mL of PEG20000, ATP [y-33P]-ATP (approximately 9 x 1005 cpm per well), protein kinase, and substrate. The reaction cocktails were incubated at 30 °C for 60 minutes. The reaction was stopped with 50 L of 2% (v/v) H3PO4, and the plates were aspirated and washed twice with 200 L of 0.9% (w/v) NaCl. 33Pi incorporation was determined using a microplate scintillation counter (Microbeta, Wallac).

Caco-2 assay (Papp A to B)

The degree of bidirectional human intestinal permeability for compounds was estimated using a Caco-2 cell permeability assay. Caco-2 cells were seeded on polyethylene membranes in 96-well plates. Growth medium was renewed every 4 to 5 days until cells formed a confluent cell monolayer. HBSS with 10 mM HEPES at pH 7.4 was used as transport buffer. Compounds were tested at 2 μM bidirectionally in duplicate. Digoxin, nadolol, and metoprolol were included as standards. Digoxin was tested at 10 μM bidirectionally in duplicate, while nadolol and metoprolol were tested at 2 μM in the A-to-B direction in duplicate. The final DMSO concentration was adjusted to less than 1% for all experiments. The plate was incubated for 2 hours in a 37°C CO2 incubator with 5% CO2 in saturated humidity. After incubation, all wells were mixed with acetonitrile containing an internal standard, and the plate was centrifuged at 4,000 rpm for 10 minutes. 100 pL of supernatant was collected from each well and diluted with 100 pL of distilled water for LC/MS/MS analysis. The concentrations of test and control compounds in the stock solution, donor solution, and receptor solution were quantified by LC/MS/MS using the peak area ratio of the analyte/internal standard. The apparent permeability coefficient Papp (cm/s) was calculated using the equation:

Papp = (dCr/dt)XVr/(AxCu)

Where dCr/dt is the cumulative concentration of the compound in the receptor chamber as a function of time (pM/s); Vr is the volume of the solution in the receptor chamber (0.075 mL on the apical side, 0.25 mL on the basolateral side); A is the surface area for transport, which is 0.0804 cm2 for the monolayer area; C0 is the initial concentration in the donor chamber (pM). The Papp scores are presented in Table 3 for the compounds.

The efflux ratio was calculated using the equation:

Efflux Ratio = Papp(BA)/ Papp(AB)

The recovery percentage was calculated using the equation:

% Recovery = 100 x [(Vr x Cr) (Vd x Cd)] / (Vd x C0)

Where Vd is the volume in the donor chambers, which are 0.075 mL on the apical side and 0.25 mL on the basolateral side; Cd and Cr are the final concentrations of the carrier compound in the donor and recipient chambers, respectively.

CYP enzyme inhibition assay

Inhibition of various CYP isoenzymes for each compound was measured using a human liver microsomal (HLM) CYP enzyme inhibition assay. Standard compounds and inhibitors were prepared in 100x working solutions. Substrates or PBS were added to the corresponding wells, followed by the addition of compounds, solvent, or positive control working solution to the corresponding wells. HLMs were added to the prewarmed (37 °C) plate and mixed with the cofactor NADPH for 10 minutes at 37 °C. The reaction was stopped by adding 400 µL of cold stop solution (200 ng/mL Tolbutamide and 200 ng/mL Labetalol in ACN). The plate was centrifuged at 4,000 rpm for 20 minutes, and the supernatant was transferred to 100 µL of HPLC water followed by LC/MS/MS analysis. The IC50s of the CYP isoenzymes for each compound were averaged and are presented in Table 3 in pM.

Measuring the metabolic stability of compounds

The metabolic stability of compounds in mouse and rat hepatocytes was determined. The half-lives of compounds are presented in Table 3 in minutes. Compounds were diluted to 5 pM in Williams E medium from 10 mM stock solutions. Aliquots of 10 pL of each compound were formed into a well of a 96-well plate, and reactions were initiated by aliquoting 40 pL of a 625,000 cell/mL suspension into each well. The plate was incubated at 37 °C with 5% CO2. At each corresponding time point, the reaction was stopped by quenching with ACN containing internal standards (IS) in a 1:3 ratio. The plates were shaken at 500 rpm for 10 min and then centrifuged at 3,220 x g for 20 min. The supernatants were transferred to another 96-well plate containing a dilution solution. The supernatants were analyzed by LC/MS/MS. The compound's half-life was estimated using the following equation:

% of Remaining Compound =

Peak Area Ratios of the Tested Compound vs. Internal Standard at End Point Peak Area Ratios of the Tested Compound vs. Internal Standard at Initial Point

hERG inhibition assay

The effects of compounds on hERG potassium channel conductance were assessed using the QPatchHTX automated patch-clamp method. Compounds were prepared in 10 mM stock solutions. CHO cells stably expressing hERG potassium channels were used for this assay. Cells were cultured in a humidified, air-controlled 5% CO2 incubator at 37°C. Stock solutions and the positive control amitriptyline were dissolved in 100% DMSO to prepare various concentrations of solution. These solutions were further diluted in extracellular solution to achieve final concentrations for the assay. The final concentration of DMSO in extracellular solution was 0.30%. The hERG QPatchHTX assay was performed at room temperature.

The following voltage command protocol was used:

• From the holding potential of -80 mV, the voltage was first increased to -50 mV for 80 ms for leak subtraction, and then to 20 mV for 4,800 ms to open hERG channels.

• After this, the voltage was reduced to -50 mV for 5,000 ms, causing a “bounce” or tail current, which was measured and collected for data analysis.

• Finally, the voltage was reduced to the holding potential of -80 mV for 3,100 ms.

For each experiment, three 5 μL additions of vehicle were applied, followed by 30 runs of the voltage command protocol over a baseline period. Then, ascending doses of each compound were added with three replicates (5 μL of compound each time). Percent control values were calculated for compounds by taking the ratio of the current response in the presence of the compound to the peak current in the presence of the vehicle control and multiplying by 100% (see Table 3).

Solubility test of compounds

The relative kinetic solubility of each compound was determined at both low (1.2) and neutral (7.4) pH. Compounds were prepared in 10 mM stock solutions. The kinetic solubility was determined by UV and calibrated using a 3-standard curve (1, 20, and 200<j>M). Compounds were allowed to stir at room temperature for 24 hours in 50 mM phosphate buffer pH 7.4, or in SGF buffer pH 1.2 at 37 °C for 24 hours. The solubilities of the compounds are reported in Table 3 in<j>M.

Kinome analysis

Recombinant kinases were produced and purified from E. coli or HEK-293 cells of strain BL21. Kinases were subsequently labeled with DNA for detection by qPCR. Streptavidin-coated magnetic beads were treated with biotinylated small molecule ligands for 30 min at room temperature to generate affinity resins for kinase assays. Ligated beads were blocked with excess biotin and washed with blocking buffer (SeaBlock with 1% BSA, 0.05% Tween 20, and 1 mM DTT) to remove unbound ligand and reduce nonspecific phage binding. Binding reactions were assembled by combining kinases, ligated affinity beads, and compounds in binding buffer (20% SeaBlock, 0.17x PBS, 0.05% Tween 20, 6 mM DTT). Compounds were prepared as 40x solutions in 100% DMSO and diluted directly into the assay. All reactions were performed in 384-well polypropylene plates in a final volume of 0.02 mL. Assay plates were incubated at room temperature with shaking for 1 hour, and affinity beads were washed with wash buffer (1x PBS and 0.05% Tween 20). Beads were then resuspended in elution buffer (1x PBS, 0.05% Tween 20, 0.5 µM non-biotinylated affinity ligand) and incubated at room temperature with shaking for 30 minutes. Kinase concentration in the eluates was measured by qPCR. Binding interaction results from the primary screening are reported as “% Ctrl,” where lower numbers indicate stronger affinity for a tested compound.

Xenograft studies in rodents

Human cancer cell line xenograft models were established in six-week-old CD-1 athymic nude mice by subcutaneous injection of 1.0–3.0 x 10 cells with or without 50% matrigel. When tumors reached an average size of 150–300 mm, mice (n = 8) were randomly assigned to treatment groups. Tumor xenografts were measured with calipers three times weekly, and tumor volume (in mm) was determined by multiplying height x width x length. For ER+ studies, 60-day-releasing pellets of 17-pestradiol were implanted subcutaneously in the left flank seven days prior to tumor inoculation. Statistical differences between treatment arms at specific time points were performed using a two-tailed, paired Student's t test. Differences between groups were considered statistically significant at p < 0.05. Compounds were formulated as 1% HEC in 25 mM phosphate buffer (pH = 2) and dosed by daily oral gavage (PO). Data were analyzed using StudyLog software from StudyDirector (San Francisco, CA).

Single-dose pharmacokinetic and saturation studies in rodents

For pharmacokinetic (PK) analysis of compounds, six-week-old, tumor-free CD-1 athymic nude mice received a single PO dose of compound followed by saphenous vein blood collection at the following post-dosing time points: 15, 30, 60, 120, 240, 480, and 1,440 minutes. No mouse was bled more than twice in the 1,440-minute period. Untreated samples were collected from vehicle control animals. For plasma preparation, whole blood was collected in EDTA-treated tubes. Cells were removed from the plasma by centrifugation for 10 minutes at 1,000-2,000 x g using a refrigerated centrifuge. The plasma fraction was extracted and stored at -80°C.

To determine the concentration at which drug exposure saturates, mice were dosed as described above with increasing concentrations of the compound covering a logarithmic concentration range (100 mg/kg to 1,000 mg/kg). Triplicate mice were used for each collection time point and dose.

To determine the amount (ng/mL) of the compound in the peripheral circulation, plasma samples were analyzed by mass spectrometry (HPLC). For this analysis, 20 pL of plasma sample was mixed with two volumes of ice-cold internal standard solution (ISS) and centrifuged at 6,100 g for 30 minutes. ISS contained acetonitrile with 100 ng/mL of the compound, 50 ng/mL of dextromethorphan, and 50 ng/mL of imipramine. Aliquots of the supernatant were transferred to an autosampler plate and diluted with two volumes of 0.2% formic acid in water. Specific analyte concentrations were determined against a standard curve (10,000 - 5 ng/mL), and mean concentrations /standard deviation were calculated.

Maximum tolerated dose studies in rodents

For maximum tolerated dose (MTD) determination studies, tumor-free CD-1 athymic nude mice were randomized into 5 treatment groups (5 mice per group) and treated with 500, 400, 300, 200, or 100 mg/kg of compound PO daily. Mice were weighed daily, and percent body weight loss was calculated relative to individual mouse body weights at the start of treatment. Studies continued for 14 days or until a group average body weight loss of >10% was observed in the animals. The MTD was determined as the highest dose at which a mean body weight loss of <10% was observed over 14 days of dosing.

Designation of sensitivity and resistance cohorts and calculation of IC50 values

Human cancer cell lines were grouped as “sensitive” or “resistant” to CDK4/6 inhibition based on whether their growth was delayed by ribociclib, palbociclib, and abemaciclib (see Table 2). These sensitive and resistant cohorts were interrogated to determine their response to each compound, and IC50s were calculated for each cell line using the same technique described above. The average IC50s for the sensitive and resistant cohorts were calculated as group geometric means. The “therapeutic window” for each compound was calculated by dividing the average IC50 for the drug-resistant group by the average IC50 for the drug-sensitive group.

Table 2. Cell line cohorts

Example 6: Single-treatment PK to identify the saturation dose

• Plasma collected at 6 time points (30 min, 60 min, 2 h, 4 h, 8 h and 24 h).

• The AUC and dose for saturation exposure were determined.

• Three mice were used per time point and the same mice were bled twice.

° 30 min and 4 h

° 60 min and 8 h

or 2 hours and 24 hours

° 9 mice per concentration

or 5 concentrations (1.78-fold dilution, 1000 mg/kg to 100)

Example 7: 14-day MTD dose de-escalation

• Started at 500 mg/kg, dose up to >0.10% BWL, then reduced to 400 mg/kg with 4 new mice, and so on.

• Abmaciclib MTD (250, 200, 150, 100 mg/kg) is also defined.

Example 8 - Selection guided by inhibitor activities

Subgenres of CDK4/6 inhibitors with desirable properties were identified using a combination of in vitro data.

In particular, the results of the assays described above (e.g., Cell Line Growth Retardation Assay, Cdk4 and 6 Enzyme Inhibition Assay, Caco-2 (PappA to B) Assay, Measurement of Metabolic Stability of Compounds and Designation of Sensitive and Resistant Cohorts and Calculation of Average IC50 Values) were used to select compounds that had defined structural and functional characteristics in the subgenera of Formula (IVa) and Formula (IVb). In particular, selected compounds were examined in sensitive and resistant cell lines, as described above. The log differences between sensitive and resistant cohorts for selected compounds are plotted in Figure 37.

In general, the Ki for CDK4 correlated with the magnitude of the therapeutic window (i.e., the difference in potency between sensitive and resistant cell line cohorts), with a lower Ki for CDK4 being associated with a wider therapeutic window. This correlation ends when the Ki for CDK4 is approximately 0.960 nM or higher.

The skilled artisan would readily recognize that the results of additional assays (e.g., CYP Enzyme Inhibition Assay, hERG Inhibition Assay, Compound Solubility Assay, Kinome Analysis, Rodent Xenograft Studies, Rodent Single Dose Pharmacokinetic and Saturation Studies, Rodent Maximum Tolerated Dose Studies, and Oral Bioavailability Assay) could be used to identify other subgenera of CDK4/6 inhibitors, or to narrow down subgenera determined using other results, e.g., the subgenera of Formulas IVa and IVb.

Table 4. PK of single-dose multi-treatment

Molecule<Dose>

(mg/kg)# of mice body weight

mouse# of dose amount of molecule<Total>A1 100.0 9 0.03 1 27.00

A1 178.0 9 0.03 1 48.06

A1 316.8 9 0.03 85.55

A1 564.0 9 0.03 1 1 152.27

A1 1003.9 9 0.03 1 271.05 583.93

A2 100.0 9 0.03 1 27.00

A2 178.0 9 0.03 1 48.06

A2 316.8 9 0.03 1 85.55

A2 564.0 9 0.03 1 152.27

A2 1003.9 9 0.03 1 271.05 583.93

weight of the number of quantity of Molecule<Dose>

(mg/kg)# mice mouse dose molecule<Total>A23 100.0 9 0.03 1 27.00

A23 178.0 9 0.03 1 48.06

A23 316.8 9 0.03 1 85.55

A23 564.0 9 0.03 1 152.27

A23 1003.9 9 0.03 1 271.05 583.93

Molecule<Dose>number of amount of(mg/kg)# mice weight of

mouse dose molecule<Total>Abemaciclib 100.0 9 0.03 1 27.00 Abemacilib 178.0 9 0.03 1 48.06 Abemacilib 316.8 9 0.03 1 85.55 Abemacilib 564.0 9 0.03 1 152.27 Abemaciclib 1003.9 9 0.03 1 271.05 583.93

Claims (13)

CLAIMS 1. A compound having the structure of Formula (IIb), (IIa), or (II): or a pharmaceutically acceptable salt thereof, in which: R1 is alkyl; and R2 is optionally substituted aminoalkyl; or R1 and R2, together with the carbon atom through which they are attached, form an optionally substituted 5- or 6-membered heterocyclic ring. 2. The compound of claim 1, wherein R1 is C1-C4 alkyl, preferably methyl or ethyl. 3. The compound of claim 1 or 2, wherein R2 is -(CH2)2N(H)(CH3), -(CH2)2N(H)(C(CH3)3), -(CH2)2N(H)(CH2CH2F), -(CH2)2N(CH3)(CH2CH2F), -(CH2)2N(CH3)2, -(CH2)2N(CH2CH3)2, -(CH2)2N(CH2CH3)2, and -(CH2CH(CH3))N(CH3)2. 4. The compound of any one of claims 1 or 2, wherein R is (CH)NR, R, and R are each independently H, alkyl, haloalkyl, alkenyl, or (CR, R, and R; respectively); R, R, and R are each independently H or alkyl; n is an integer having a value of 1 or 2; and m is an integer having a value of 2 to 5. 5. A compound of claim 1 selected from: or a pharmaceutically acceptable salt thereof. 6. The compound of claim 1, having the structure of Formula (IVa): or a pharmaceutically acceptable salt thereof, wherein: X' is fluoro; R1' is C1-C3 alkyl; R2' is (CR2c2)nNR2a'R2b'; R2a' is H, lower alkyl, or haloalkyl; R2b' is H, lower alkyl, or haloalkyl; each R2c' is independently H or alkyl; n' is an integer having a value of 1 or 2; and wherein the compound has a CDK4 Ki of about 0.960 nM or less. 7. The compound of claim 6, wherein the compound has an average IC50 of 150 nM or less for the drug-sensitive cell lines in the following table 8. The compound of claim 6, wherein the average IC50 of the compound for the drug-sensitive cell lines in the following table: It is at least approximately 5 times more potent than the compound's average IC50 for drug-resistant cell lines in the following table: 9. The compound of any of claims 6-8, wherein the compound has an apparent permeability coefficient (Papp) score for transport of the test substance from the apical to the basolateral side (A to B) of about 0.07 or greater. 10. The compound of any of claims 6-9, wherein the compound has a half-life of about 25 minutes or greater. 11. The compound of claim 6, wherein the compound is selected from: or a pharmaceutically acceptable salt thereof. 12. A pharmaceutical composition comprising a compound of any one of claims 1-11 and a pharmaceutically acceptable diluent or excipient. 13. A compound selected from any one of claims 1-11, or a pharmaceutically acceptable salt thereof, for use in the treatment of cancer in a subject in need thereof, or for use in sensitizing cancer and/or tumor cells in a subject in need thereof to a chemotherapeutic agent or radiation.